Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media

ABSTRACT

Methods and apparatus are presented that facilitate electrophoresis of prior-cast, hydratable separation media, usefully immobilized pH gradient (IPG) strips. The method exploits the swelling of prior-cast, hydratable separation media upon rehydration to help lodge the media in an enclosing member that permits spaced electrical communication with the enclosed separation media. The electrical communication permits a voltage gradient to be established in the enclosed separation medium sufficient to effect separation of analytes therein. Cassettes, buffer cores, electrophoresis systems and kits are presented for effecting the methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisionalapplication serial No. 60/390,259, filed Jun. 18, 2002, and is acontinuation-in-part of U.S. application serial No. 10/102,188, filedMar, 18, 2002, which claims the benefit of U.S. provisional applicationserial No. 60/290,464, filed May 10, 2001, the disclosures of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to methods and apparatus forelectrophoresis of prior-cast hydratable separation media. Inparticular, the invention relates to methods, cassettes, buffer cores,and systems useful for conducting isoelectric focusing using immobilizedpH gradient (IPG) strips.

BACKGROUND OF THE INVENTION

[0003] For over thirty years, isoelectric focusing (IEF) has served as aprimary tool for analyzing proteins present in complex admixture, suchas proteins present in biological samples.

[0004] In isoelectric focusing, proteins are driven by an appliedelectric field through a pH gradient typically established in a supportmatrix, such as a gel. Proteins migrate until the isoelectric point (pI)of the protein coincides with the local pH; at that point, the proteinno longer bears net charge and ceases to migrate, becoming focused at apoint that is characteristic of the protein.

[0005] As originally described, the pH gradient for IEF was establishedand sustained in the gel matrix by mobile carrier ampholytes (CA). Gelstypically would be polymerized in the presence of a population of CAhaving a range of charge characteristics; upon application of a voltagegradient, the various species of CA would align themselves in the matrixto establish a pH gradient across the gel.

[0006] Although IEF with CA has proven tremendously useful, it was soondiscovered that pH gradients created by CA were susceptible to titrationby atmospheric carbon dioxide, leading to the migration of CA towardsthe cathode and destruction of the pH gradient over time, a phenomenontermed cathodic drift.

[0007] Cathodic drift can be reduced by casting IEF gels in enclosedtubes, thus limiting exposure to atmospheric CO₂. However, the tubetraps prepolymer component impurities in the matrix duringpolymerization, interfering with separation. Furthermore, the tubeformat presents difficulties when a second dimension of separation, suchas fractionation by size, is desired.

[0008] In a different approach to the problem of cathodic drift,Bjellqvist and colleagues immobilized the pH gradient in the supportmatrix, an approach now termed immobilized pH gradient (IPG) isoelectricfocusing. See Bjellqvist et al., J. Biochem. Biophys. Methods6(4):317-39 (1982); Righetti et al., Trends Biochem. Sci. 13(9):335-8(1988); Righetti et al., Methods Enzymol. 270:235-55 (1996); U.S. Pat.No. 4,130,470; and Righetti, Immobilized pH Gradient: Theory andMethodology, (Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 20), Elsevier Biomedical Press, LTD, Netherlands (ASIN:0444813012). Two-dimensional electrophoresis, with IPG IEF followed bysize fractionation, soon followed. Gorg et al., Electrophoresis9(9):531-46 (1988).

[0009] IPG not only reduced the problem of cathodic drift, but alsoproved useful in reducing interference from prepolymer componentimpurities, since the IPG strip's plastic backing imparts sufficientstructural resilience to the gel as to permit the gel to be washedbefore use. The increased resilience also permits the gels to be storedin dehydrated form before use. Dehydrated IPG strips are today sold in avariety of pH ranges and a variety of separation lengths by a number ofvendors (e.g., Immobiline DryStrip Gels, Amersham Biosciences,Piscataway, N.J., USA; ReadyStrip IPG, Bio-Rad Laboratories, Hercules,Calif., USA).

[0010] Problems remain, however.

[0011] Although immobilization of the gradient-forming ampholytesprevents cathodic drift, the charge-bearing immobilized moieties(immobilines) remain susceptible to titration by atmospheric CO₂. CO₂titration is exacerbated by the fact that the separation medium of IPGstrips is directly exposed to air on at least one side. Direct exposureto air also leads to possible dehydration of the matrix, with possiblesalt crystallization, during electrophoresis.

[0012] These problems have been addressed in part by a methodological,rather than structural, solution: plastic-backed IPG strips aretypically electrophoresed under an occlusive oil layer, which bothexcludes air and retards evaporation.

[0013] Use of an occlusive liquid oil layer presents its owndifficulties, however. Principal among these is the requirement thatelectrophoresis be performed with the IPG strip maintained in ahorizontal orientation. The obligate horizontal orientation precludesuse of the smaller-footprint, vertical electrophoresis devices typicallyused for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), such asthose described in Tippins et al., U.S. Pat. No. 5,888,369. In addition,the use of oil requires deft manual technique and proves time-intensive.

[0014] Wiktorowicz et al., U.S. Pat. No. 6,013,165, describe anapparatus in which immobilized pH gradient isoelectric focusing can beperformed without use of a liquid oil layer. A continuous pKa gradientis immobilized on at least one of the major opposing surfaces of acavity formed between two plates. The cavity, which can be furthersegmented into parallel channels, is then filled with a flowableseparation medium. Electrophoresis is preferably conducted with theassembly oriented horizontally to minimize convection currents in theflowable separation medium. The apparatus does not readily permitinsertion of prior-cast hydratable separation media, such as commercialIPG strips, nor does it readily permit electrophoresis in the verticaldimension.

[0015] There thus exists a need in the art for methods and apparatusthat allow IPG strips, and other prior-cast hydratable separation media,to be electrophoresed without requiring contact with an occlusive fluidoil layer. There further exists a need in the art for methods andapparatus that allow IPG strips, and other prior-cast hydratableseparation media, to be electrophoresed in a vertical orientation.

SUMMARY OF THE INVENTION

[0016] The present invention solves these and other needs in the art byproviding methods, apparatus, and kits for electrophoresis of prior-casthydratable separation media that obviate the use of an occlusive oillayer, thereby obviating the requirement that electrophoresis beperformed in the horizontal orientation.

[0017] The present invention is based, in part, upon the discovery thatthe swelling that attends rehydration of prior-cast hydratableseparation media can be exploited to help lodge such media in anenclosure that permits spaced electrical communication with the enclosedseparation medium. The spaced electrical communication makes it possibleto apply a voltage gradient to the prior-cast hydratable separationmedia while the medium is otherwise enclosed, permitting electrophoresisto be conducted within a cassette.

[0018] Enclosed, the separation medium's contact with air issubstantially reduced. In cases in which the prior-cast hydratableseparation medium is an IPG strip, the reduction in air contact obviatesthe prior art requirement for occlusive contact with a fluid oil layerduring immobilized pH gradient isoelectric focusing.

[0019] Enclosed, and lacking an attendant fluid oil layer, theprior-cast separation medium can be electrophoresed in any physicalorientation. In cases in which the prior-cast hydratable separationmedium is an IPG strip, relaxation of the prior-art requirement forhorizontal electrophoresis makes it newly possible to perform IPGelectrophoresis using the widely distributed, small footprint, verticalelectrophoresis gel boxes presently used to perform SDS-PAGE.

[0020] The invention is further based upon novel apparatus designs thatminimize the resistance between power supply and gel; the reduction inparasitic system impedances permits separations, particularlyisoelectric focusing in IPG strips, to be performed using lower voltagesfor reduced times.

[0021] Thus, in a first aspect, the invention provides a method forperforming electrophoresis, comprising: hydratingly lodging a prior-casthydratable electrophoretic separation medium within an enclosing memberthat permits spaced electrical communication with the enclosed medium;and then using the spaced electrical communication to establish avoltage gradient in the enclosed separation medium sufficient to effectelectrophoretic separation of analytes therein.

[0022] In one embodiment, the method further comprises the antecedentstep of inserting the prior-cast hydratable electrophoretic separationmedium in its dehydrated state into the enclosing member. In anotherembodiment, the method further includes a later step of removing theprior-cast hydratable electrophoretic separation medium from theenclosing member. The medium once removed can be used, for example, toapply the one-dimensionally fractionated sample to a gel to effect asecond dimension of separation.

[0023] In some embodiments, the step of hydratingly lodging comprises:contacting the dehydrated prior-cast hydratable electrophoreticseparation medium with an aqueous solution, often an aqueous solutionthat includes the sample to be fractionated.

[0024] The methods of the present invention are particularly useful inperforming isoelectric focusing using immobilized pH gradient strips.Thus, the prior-cast hydratable electrophoretic separation medium usedin the practice of the present invention can usefully have animmobilized pH gradient.

[0025] As described above, the methods of the present invention includethe use of an enclosing member that has (i) means for hydratinglylodging a prior-cast electrophoretic separation medium therewithin, and(ii) means for spaced electrical communication with the enclosedseparation medium, wherein the spaced electrical communication means canbe used to apply a voltage gradient to the enclosed medium sufficient toeffect electrophoretic separation of analytes present therewithin.

[0026] Thus, in another aspect, the invention provides a cassette forperforming electrophoresis, comprising: means for hydratingly lodging aprior-cast electrophoretic separation medium within an enclosing member;and means for spaced electrical communication with the enclosed medium,wherein the spaced electrical communication means can be used toestablish a voltage gradient in the separation medium sufficient toeffect electrophoretic separation of analytes therein.

[0027] In certain embodiments, the cassette of the present inventioncomprises: a form-retaining member, and at least one channel, whereinthe form-retaining member imparts dimensional integrity to the channelor channel(s). In typical embodiments, the cassette includes a pluralityof such channels.

[0028] Each channel present in the cassette and useful for performingthe methods of the present invention has a first channel entry, a secondchannel entry, and a cavity therebetween, the channel cavity being sodimensioned as to permit insertion of a hydratable prior-castelectrophoretic separation medium in its dehydrated state and lodginglyenclose the strip in its rehydrated state. The first and second channelentries permit spaced electrical communication with the channel cavity;the spaced electrical communication permits current to be flowed throughthe channel cavity.

[0029] In some embodiments, the form-retaining member contributes theentire circumferential wall of the cavities of the channels. In other,multilaminate embodiments, the cassette further comprises a laminatecover; the laminate cover adheres directly or indirectly to theform-retaining member and contributes at least part of thecircumferential wall of said channels. In these latter embodiments, theadherence of the laminate cover to the form-retaining member istypically reversible.

[0030] In other embodiments, the cassette further comprises a firstwell-forming member, which adheres directly or indirectly to theform-retaining member, and which defines fluid reservoirs at a pluralityof first channel entries. Usefully, the cassette can further comprise asecond well-forming member, the second well-forming member adhering,directly or indirectly, to the form-retaining member and defining fluidreservoirs at a plurality of second channel entries. When present, thewell-forming members can usefully be reversibly adherent to theform-retaining member.

[0031] In one series of related embodiments, the first and secondchannel entries for each of the channels permit electrical communicationwith the intervening channel cavity through a common surface of thecassette. In another series of related embodiments, the first and secondchannel entries permit electrical communication with their interveningcavity through separate surfaces of the cassette. These two mutuallyexclusive geometries call for different electrode geometries, and thusdifferent electrophoresis buffer cores, to complete the circuitsrequired for electrophoresis.

[0032] The prior-cast hydratable electrophoretic separation medium canbe provided by the user, can be included within one or more channels ofthe cassette without requirement for user insertion thereof, or can beprovided separately packaged with the cassette in a kit.

[0033] As to the latter, it is another aspect of the present inventionto provide kits for facilitating electrophoresis of prior-casthydratable electrophoretic separation media. The kits typically comprisea cassette of the present invention and at least one prior-casthydratable electrophoretic separation medium suitably dimensioned as tobe hydratingly lodgeable in said cassette.

[0034] In some embodiments, the kit includes a cassette and at least oneconductive wick for use therewith; often, in such kits, a sufficientnumber of wicks are provided to facilitate both anodic and cathodicconnections with the cassette.

[0035] The cassettes of the present invention can be used to effectvertical electrophoresis of prior-cast hydratable separation media,usefully in the buffer tanks that are commonly used, with buffer cores,for SDS-PAGE electrophoresis. In cassette embodiments in which the firstand second channel entries open to separate surfaces of the cassette,buffer cores presently used for SDS-PAGE electrophoresis can be used. Incassette embodiments in which the first and second channel entries opento the same surface of the cassette, alternative buffer core geometriesare required.

[0036] Thus, it is another aspect of the present invention to provide abuffer core for vertical electrophoresis of pre-cast hydratableelectrophoretic separation media, comprising: a substantially inflexibleframe, an anode, and a cathode in spaced relationship to the anode. Thebuffer core frame has a first cassette engagement face and a secondcassette engagement face. Operational engagement of a first and secondcassette to the respective first and second frame engagement facescreates a chamber internal to the frame that is sealed on 5 sides. Thecathode and anode are each in electrical communication with the interiorof the internal chamber, and operational engagement of a first andsecond cassette to the respective first and second frame engagementfaces causes spaced contact of the anode and cathode to the surface ofat least one cassette that engages the frame engagement surface,allowing electrophoresis of prior-cast hydratable separation mediaenclosed therein.

[0037] The cassette and buffer core system of the present inventionreduces the resistance between power supply and gel, permittingelectrophoretic separation using lower voltages, for shorter times, fora lower volt-hour total.

[0038] Thus, in another aspect, the invention provides a system for lowresistance electrophoresis of analyte samples in prior-cast, hydratableseparation media strips. The system comprises means for enclosing aplurality of strips and means responsive to an external compressiveforce for effecting spaced electrical communication by a single anodeand single cathode simultaneously with each of said enclosed strips.

[0039] The enclosing means permits spaced electrical communicationseparately with each of the enclosed strips through respective first andsecond entries. The electrical communication means is capable ofdistributing an external compressive force to urge the anode and thecathode toward the enclosing means with greater pressure at the firstand second entries than elsewhere on the enclosing means.

[0040] The electrical communication means may comprise an anode support;a cathode support; an anode; and a cathode. The supports in theseembodiments discontinuously distribute an external compressive force tothe anode and cathode to urge the anode and cathode toward the enclosingmeans with greater pressure at the first and second entries thanelsewhere on the enclosing means. In certain embodiments, the anodesupport makes discontinuous contact with the anode and the cathodesupport makes discontinuous contact with the cathode.

[0041] In typical embodiments of the system of this aspect of theinvention, the enclosing means is capable of hydratingly lodging stripsthere within.

[0042] The system provides a substantially reduced resistance pathwaybetween power supply and gel than do prior art devices forelectrophoresis of prior-cast, hydratable, separation media, such as IPGstrips.

[0043] The system of this aspect of the invention may, for example, becapable of effecting electrophoretic separation, including isoelectricfocusing in IPG strips, with application of a maximum of 3000 or fewervolts, 1500 or fewer volts, even 500 or fewer volts. The system may becapable of effecting electrophoretic separation, including isoelectricfocusing in IPG strips, with application of fewer than 2000 volt-hours,even as few as 1500 volt-hours. The system of this aspect of theinvention may be capable of effecting electrophoretic separation,including isoelectric focusing in IPG strips, in fewer than 6 hours, 5hours, even 4 hours or less.

[0044] In another aspect, the invention provides a method for lowresistance electrophoresis of analyte samples in prior-cast, hydratableseparation media strips.

[0045] The method comprises hydratingly lodging at least one stripwithin an enclosing member that permits separate, spaced, electricalcommunication with each of a plurality of enclosed strips throughrespective first and second entries; applying a sample containingprotein analytes to the enclosed strip; forcibly urging an anode and acathode toward the enclosing member to effect simultaneous spacedelectrical communication with each of the enclosed strips, wherein theforce urging the anode and the cathode toward the enclosing member isdistributed to create greater contact pressure at the first and secondentries than elsewhere on the enclosing member; and then applyingelectrical potentials to the anode and cathode at a potential differenceand for a time sufficient to effect electrophoretic separation ofanalytes in the enclosed strips.

[0046] In some embodiments, sample is applied during lodging of thestrip in said enclosing member.

[0047] The method provides a substantially lower resistance pathwaybetween power supply and separation media than is found in the priorart.

[0048] Accordingly, effective separation, including isoelectric focusingin IPG strips, may be obtained in the methods of this aspect withapplication of a maximum of 3000 or fewer volts, 1500 or fewer volts,even 500 or fewer volts. The methods may effect electrophoreticseparation, including isoelectric focusing in IPG strips, withapplication of fewer than 2000 volt-hours, even as few as 1500volt-hours. The methods may be capable of effecting electrophoreticseparation, including isoelectric focusing in IPG strips, in fewer than6 hours, 5 hours, even 4 hours or less.

[0049] In the methods of this aspect of the invention, the potentialdifference may be applied in a plurality of ramped voltage steps, in aplurality of stepped voltage steps, or at a constant voltage level.

[0050] The strip may usefully be an IPG strip.

[0051] In a related aspect, the invention provides improved methods ofelectrophoresis using prior-cast, hydratable, separation media strips,wherein the improvement comprises spacedly contacting the strips with ananode and cathode with resistance between power supply and gelsufficiently low as to permit electrophoretic separation with a maximumapplied voltage of no more than 3000 volts, no more than 1500 volts,even no more than 500 volts.

[0052] In various embodiments of the methods of this aspect of theinvention, the strip is an immobilized pH gradient (IPG) strip, and theresistance is sufficiently low as to permit isoelectric focusing withapplication of fewer than 2000 nominal volt-hours, even as few as 1500nominal volt-hours.

[0053] In yet another aspect, the invention provides a buffer coredevice for forcibly urging an anode and a cathode into simultaneousspaced electrical communication with a plurality of prior-casthydratable separation media strips enclosed within means that permitspaced electrical communication separately with each of the enclosedstrips through respective first and second entries.

[0054] The device comprises a substantially inflexible frame; an anodesupport; a cathode support; an anode; and a cathode. The anode supportand the cathode support are spacedly fixed to the frame and are capableof distributing an external compressive force respectively to thecathode and the anode to urge the cathode and the anode toward theenclosed strips with greater contact pressure at the first and secondentries than elsewhere on the enclosing means.

[0055] The anode support may make intermittent contact with the anodeand the cathode support may make intermittent contact with the cathode.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The above and other objects and advantages of the presentinvention will be apparent upon consideration of the following detaileddescription taken in conjunction with the accompanying drawings, inwhich like characters refer to like parts throughout, and in which:

[0057]FIG. 1A is a front perspective view of one embodiment of acassette of the present invention;

[0058]FIG. 1B is a front perspective view of another embodiment of acassette of the present invention;

[0059]FIG. 1C is a front perspective view of the embodiment of FIG. 1B,rendered as opaque;

[0060]FIG. 2 is a front perspective view of a cassette of the presentinvention with an IPG strip inserted into one of six available channels;

[0061]FIG. 3A is a front perspective view of a cassette of the presentinvention, with well-forming member removed, prior to application of aconductive wick;

[0062]FIG. 3B is a front perspective view of a cassette of the presentinvention with a first conductive wick contacting the anodic end of IPGstrips present in three of six available channels and a secondconductive wick contacting the cathodic end of the three IPG strips;

[0063]FIG. 3C is a back perspective view of an embodiment of a cassetteof the present invention, particularly showing a recessed region thatfacilitates heat dissipation during electrophoresis;

[0064]FIG. 3D is a back perspective view of another embodiment of acassette of the present invention, particularly showing a plurality ofrecessed regions that facilitate heat dissipation duringelectrophoresis;

[0065]FIG. 4 is an exploded side perspective view of a multilaminatecassette of the present invention;

[0066]FIG. 5 is an exploded side perspective view of a loading wellassembly of a cassette of the present invention;

[0067]FIG. 6A is a front perspective view of a buffer core of thepresent invention, without anode electric wire or cathode electric wire,with gasket;

[0068]FIG. 6B is a front perspective view of a buffer core of thepresent invention (front) operationally aligned to contact its anode andcathode electrodes respectively to anodic and cathodic wicks of acassette of the present invention (rear);

[0069]FIG. 6C is a front perspective view of the buffer core andcassette of FIG. 6B in operational contact with one another;

[0070]FIG. 6D is a front perspective view of a buffer core inoperational contact with two cassettes of the present invention;

[0071]FIG. 6E shows a buffer core of the present invention, withcassettes of the present invention operationally engaged thereupon,further engaged in an electrophoresis chamber;

[0072]FIG. 7A is a front view of a cassette of the present invention inwhich channel entries open through opposite surfaces of the cassette;

[0073]FIG. 7B is a side view of the cassette of FIG. 7A;

[0074]FIG. 7C is an exploded perspective view of two cassettes as shownin FIGS. 7A and 7B showing their operational relationship to a prior artbuffer core;

[0075]FIG. 7D is a perspective view of the cassettes of FIGS. 7A and 7Bin operational contact with a prior art buffer core;

[0076]FIG. 8 shows IPG strips after electrophoresis in channels of thestated internal dimensions;

[0077]FIG. 9 plots measured currents through IPG strips run, forcomparison, in the cassette of the present invention using the buffercore of the present invention, and in a commercially availablehorizontal flat bed apparatus, using identical power supplies withidentical voltage programs;

[0078]FIG. 10 plots currents measured through IPG strips, and voltagesreported by two power supplies, using the cassette and buffer core ofthe present invention, with focusing performed using a stepped voltageprofile; and

[0079]FIG. 11 illustrates an electrophoresis tank cover useful forapplying voltage to the cassette and buffer core of the presentinvention.

DETAILED DESCRIPTION

[0080] The present invention is based, in part, upon the discovery thatthe swelling that attends rehydration of prior-cast hydratableseparation media can be exploited to help lodge such media in anenclosure that permits spaced electrical communication with the enclosedseparation medium. The spaced electrical communication makes it possibleto apply a voltage gradient to the prior-cast hydratable separationmedia while the medium is lodged within the enclosing member.

[0081] Enclosed, the separation medium's contact with air issubstantially reduced. In cases in which the prior-cast hydratableseparation medium is an IPG strip, the reduction in air contact obviatesthe prior art requirement for occlusive contact with a fluid oil layerduring immobilized pH gradient isoelectric focusing.

[0082] Enclosed, and lacking an attendant fluid oil layer, theprior-cast separation medium can be electrophoresed in any physicalorientation. In cases in which the prior-cast hydratable separationmedium is an IPG strip, relaxation of the prior-art requirement forhorizontal electrophoresis makes it newly possible to perform IPGelectrophoresis using the widely distributed, small footprint, verticalelectrophoresis gel boxes presently used to perform SDS-PAGE.

[0083] In a first aspect, therefore, the invention provides a method forperforming electrophoresis, particularly for performing electrophoresisusing prior-cast, hydratable separation media. As used herein, the term“electrophoresis” explicitly includes isoelectric focusing.

[0084] In a first step, the method comprises hydratingly lodging aprior-cast hydratable electrophoretic separation medium within anenclosing member that permits spaced electrical communication with theenclosed media. In a second step, the spaced electrical communication isused to apply a voltage gradient to the enclosed medium sufficient toeffect electrophoretic separation of analytes therein.

[0085] As used herein, the phrase “prior-cast electrophoretic separationmedium” (and equivalently, “prior-cast separation medium”) refers to anelectrophoretic separation medium, typically a polymeric gel, that hasfirst been solidified, or gelled, elsewhere than in the enclosing memberin which electrophoresis is to be performed.

[0086] Electrophoretic separation media, and methods of preparing,casting, and performing electrophoresis using electrophoretic media, arewell known in the analytical arts, and need not be detailed here. See,e.g., Rabilloud (ed.), Proteome Research: Two-Dimensional GelElectrophoresis and Identification Methods, Springer Verlag, 2000 (ISBN:3540657924); Westermeier, dimension substantially greater than a seconddimension—such dimensions are not required for practice of the presentinvention. Nonetheless, for ease of description, all prior-castelectrophoretic separation media useful in the practice of the presentinvention are referred to in the alternative herein as “strips”.

[0087] A “prior-cast hydratable electrophoretic separation medium” is aprior-cast electrophoretic medium that can be dehydrated and that, afterrehydration, has retained sufficient structural integrity to permitelectrophoretic separation of analytes there within.

[0088] Neither complete removal of moisture, during dehydration, norcomplete saturation with liquid, during rehydration, is required orintended. It suffices for practice of the present invention that theprior-cast, hydratable, electrophoretic separation medium swelldetectably after contact in its dehydrated state with an aqueoussolution (“aqueous buffer”, “buffer”).

[0089] Typically, the prior-cast hydratable electrophoretic separationmedium will swell at least about 5% in volume, often at least about 10%,15%, 20%, even at least about 25%, 30%, 40% or more in volume uponcontact with an aqueous buffer. The volume increase can be manifest inall three dimensions or, when the separation medium is backed with aninextensible layer, principally in one or in two dimensions. The volumeincrease can occur over a period of minutes or, in the case of IPGstrips, more typically over a period of hours.

[0090] The degree of swelling is sufficient if the prior-cast,hydratable, electrophoretic separation medium swells sufficiently uponcontact with an aqueous solution Electrophoresis in Practice, 2nd ed.,John Wiley & Sons, 2000 (ISBN 3527300708); B. D. Hames et al. (eds.),Gel Electrophoresis of Proteins, 3rd ed., Oxford University Press, 1998)(ISBN 0199636419); and Jones, Gel Electrophoresis: Nucleic Acids:Essential Techniques, (John Wiley & Son Ltd. 1996) (ISBN 0471960438),the disclosures of which are incorporated herein by reference in theirentireties.

[0091] Although polyacrylamide (that is, a polymerization product ofacrylamide monomer crosslinked with N,N′-methylenebisacrylamide) andagarose are the two polymeric gels most commonly used in electrophoresistoday, the present invention proves useful in electrophoresing a farwider variety of polymeric gels.

[0092] Because the gel is first solidified, or gelled, elsewhere than inthe enclosing member in which electrophoresis is to be performed, the“prior-cast electrophoretic separation medium” used in the presentinvention must have sufficient structural resiliency to be transferredor released from its casting mold and thereafter lodged within theenclosure of the present invention.

[0093] Typically, such structural resiliency will be imparted to theseparation medium by the adherence thereto or incorporation therein of alayer or lamina of another material, such as plastic. Such layers areknown in the art, and include, e.g., polyester film backings, as arefound in commercial IPG strips, and polyester mesh fabric, which can beincorporated into the separation medium.

[0094] Although the “prior-cast electrophoretic separation medium” usedin the present invention is typically fashioned as a strip—that is, witha first (“aqueous buffer”, “buffer”) as to permit hydratable lodging inan enclosing member.

[0095] By “hydratable lodging” is intended that the prior-cast,hydratable separation medium be insertable into an enclosing member inits dehydrated state, and that it become lodged in the enclosing memberin its rehydrated state.

[0096] Although the strip must be “insertable” in its dehydrated state,the strip need not necessarily be removable from the enclosing member inits dehydrated state.

[0097] The rehydrated prior-cast hydratable separation medium is said tobe “lodged” in the enclosing member (equivalently, “lodgingly enclosed”therein) when two conditions are met. First, the strip remains withinthe enclosing member when the enclosing member is brought into verticalorientation. Second, when the enclosing member is brought into verticalorientation, at least 50% of the separation medium is precluded fromdirect communication with ambient atmosphere. Furthermore, althoughfrictional and surface tension forces between the rehydrated separationmedium and the enclosing member can contribute to the strip's lodgingtherein, it is not intended that such frictional or surface tensionforces be sufficient in themselves to effect lodging of the strip withinthe enclosing member.

[0098] The enclosing member will be sufficiently form-retaining as to beable to maintain dimensional integrity when maintained in contact with aprior-cast, hydratable separation medium that is swelling. In certainembodiments described in detail below, the enclosing member is acassette having a form-retaining channel cavity within which theprior-cast, hydratable separation medium is engaged.

[0099] The enclosing member further permits spaced electricalcommunication with the enclosed prior-cast hydratable separation medium.Communication can be direct, as by through-passage of anode and cathodeelectrodes, or indirect, as by passage of current through anintermediate polymer layer or wick, as will be further discussed below.

[0100] After the prior-cast hydratable electrophoretic separation mediumis lodged in the enclosing member, the spaced electrical communicationis used to apply a voltage gradient sufficient to effect electrophoreticseparation of analytes therein.

[0101] Although described particularly herein as application of avoltage gradient to the separation medium, it is understood that currentis thereby caused to flow through the separation medium, and that themethod could equally be described as flowing current through theseparation medium.

[0102] The electrical parameters to be used depend upon the compositionand dimensions of the enclosed electrophoretic medium, the compositionof the sample, the composition of the rehydration solution, the type ofdesired separation, and the method by which spaced electrical contact ismade with the enclosed separation medium, and can readily be determinedempirically by routine experimentation. Particular electrical parametersfor isoelectric focusing using cassette-immobilized IPG strips and abuffer core adaptor of the present invention are further describedherein below.

[0103] Returning to the method in more detail, the prior-cast hydratableelectrophoretic separation medium is typically inserted by the user inits dehydrated state in the enclosing member.

[0104] By way of example, in embodiments further described below, theprior-cast hydratable separation medium, such as an IPG strip, ismovably inserted by hand into a channel cavity present within theenclosing member. As another example, where the enclosing member ishinged, or otherwise reversibly separable, the prior-cast hydratableseparation medium, such as an IPG strip, is movably inserted by handinto a depression, with the channel cavity thereafter completed byclosing the member.

[0105] Although typical, movable insertion of the dehydrated strip intothe enclosing member is not always required. For example, the dehydratedstrip can be earlier-inserted during manufacture of the enclosingmember, obviating insertion of the dehydrated prior-cast separationmedium into the enclosing member by the user.

[0106] The dehydrated separation medium is then contacted with anaqueous solution.

[0107] The composition of the rehydration solution will depend upon thecomposition of the sample and separation medium and the intendedelectrophoretic procedure, and its choice will thus depend on factorsthat are well known in the electrophoretic arts.

[0108] For example, where the prior-cast hydratable separation medium isa commercial IPG strip, such as an Immobiline DryStrip (AmershamBiosciences, Piscataway, N.J., USA), the rehydration solution canusefully include urea, non-ionic or zwitterionic detergents,dithiothreitol (DTT), dye, and a carrier ampholyte mixture suited to thepH range of the IPG strip. Carrier ampholyte mixtures for use in suchrehydration solutions are available commercially (e.g., IPG Buffer pH3.5-5.0, cat. no. 17-6002-02; IPG Buffer pH 4.5-5.5, cat. no.17-6002-04; IPG Buffer pH 5.0-6.0, cat. no. 17-6002-05; IPG Buffer pH5.5-6.7, cat. no. 17-6002-06; IPG Buffer pH 4-7, cat. no. 17-6002-86;IPG Buffer pH 6-11, cat. no. 17-6002-78; IPG Buffer pH 3-10 NL, cat. no.17-6002-88; IPG Buffer pH 3-10, cat. no. 17-6002-87, all from AmershamBiosciences, Piscataway, N.J., USA).

[0109] The rehydration solution can also advantageously include thesample intended to be separated in the prior-cast hydratable separationmedium.

[0110] For example, in cases in which the prior-cast hydratableelectrophoretic separation medium is an IPG strip, the sample to beseparated can be a mixture of proteins, such as those from a biologicalsample, and can usefully be or have been denatured, as by chaotropes,reducing agents, and detergents. In cases in which the separation mediumis other than an immobilized pH gradient strip, the sample can includeother types of macromolecules, such as nucleic acids.

[0111] The methods of the present invention can include the later stepof removing the prior-cast hydratable separation medium from theenclosing member after electrophoresis. The method of removal willdepend on the structure of the enclosing member, as will be furtherdescribed below. As an alternative to removal, the separation medium incertain embodiments of the methods of the present invention can befurther analyzed within the enclosing member, such as by staining anddrying.

[0112] As described above, the methods of the present invention includethe use of an enclosing member that has (i) means for hydratinglylodging a prior-cast electrophoretic separation medium therewithin and(ii) means for spaced electrical communication with the enclosedseparation medium, wherein the spaced electrical communication means canbe used to apply a voltage gradient to the enclosed separation mediumsufficient to effect electrophoretic separation of analytes presenttherewithin.

[0113] It is, therefore, another aspect of the present invention toprovide an enclosing member useful in the practice of the methods of thepresent invention, which enclosing member is hereinafter called a“cassette”.

[0114] FIGS. 1A-1C are front perspective views of embodiments of acassette of the present invention.

[0115] Cassette 100 comprises form-retaining member 10 and at least onechannel 12 (in the embodiments shown in FIGS. 1A and 1B, cassette 100has six substantially parallel channels 12, although fewer or greaternumbers can be present). Form-retaining member 10 imparts dimensionalintegrity to prior-formed channels 12. Channels 12, although present,are not visible in FIG. 1C, rendered as fully opaque.

[0116] Referring again to FIG. 1A, channel 12 has first channel entry 14and second channel entry 16 and cavity 18 therebetween. Cavity 18 ofchannel 12 is so dimensioned as to movingly engage a prior-casthydratable electrophoresis medium (“strip”), such as an IPG strip, inits dehydrated state, and to lodgingly enclose the strip after hydrationthereof.

[0117] First channel entry 14 and second channel entry 16 permitelectrical communication with cavity 18, and thus define a channelcurrent flow axis through cavity 18. In certain embodiments of cassette100 particularly designed for use with buffer cores of the prior art(see below), the channel current flow axis is in a plane substantiallyparallel to a substantially planar first surface of form-retainingmember 10.

[0118] To use cassette 100 in the methods of the present invention,rehydratable electrophoresis strip 20, such as an IPG strip, is insertedin its dehydrated state into channel 12, typically through entry 14 orentry 16. In alternative embodiments, strip 20 has been prior-insertedinto cassette 100, either by the user or by the manufacturer thereof.

[0119] Strip 20 is rehydrated within channel 12 by application of arehydration solution, optionally containing the sample to befractionated.

[0120] Rehydration solution is typically dispensed into channel 12 priorto insertion of strip 20, since insertion of strip 20 into channel 12 isfacilitated by wetting of the interior of channel 12. Strip 20 can,however, be prior-inserted into channel 12, with rehydration solutionthereafter applied at either or both of entries 14 and 16. For samplesrequiring long rehydration times, entry 14, entry 16, or both can besealed—e.g. with tape or cover slip—to prevent evaporation and theaccidental discharge of rehydration solution.

[0121] Upon rehydration, strip 20 becomes lodged in cavity 18 of channel12, at least in part due to swelling of the separation medium. Strip 20is thereafter not readily removed from channel 12 without expansion ofcavity 18, as further described below.

[0122] If the sample to be electrophoretically fractionated is notincluded in the rehydration solution, sample is then applied at entry14, entry 16, or both with the cassette oriented horizontally to retainsample, and allowed to enter the separation medium. Alternatively,sample can be prior-absorbed into a wick which is then inserted intoentry 14, entry 16, or both, from which wick the sample then enters theseparation medium. As further described below, sample entry can befacilitated by application of electrical current.

[0123] Electrophoresis is then performed by applying a voltage gradientto strip 20, causing current to flow along the channel current flowaxis.

[0124] Thereafter, strip 20 is typically removed from channel 12 forfurther processing, such as staining and/or contacting of strip 20 (or aportion thereof) to a gel to effect separation along a second dimension.Removal is typically effected by expansion of cavity 18 using a methodappropriate to the composition of cassette 100; for example, inembodiments of cassette 100 in which one or more laminae contribute tothe circumferential walls of cavity 18, removal can be effected bypeeling of the laminae, thus opening channel 12. For certain purposes,further processing can be effected within channel 12.

[0125] Returning to FIG. 1A, form-retaining member 10 is constructed ofform-retaining nonliquid materials. Preferred materials are those thatare readily machined, molded, or etched, that are chemicallycompatible—that is, do not suffer substantial degradation uponcontact—with electrophoretic buffer systems, that do not appreciablybind or impede the transport of analytes through the enclosed gel, andthat provide a vapor gas barrier. Usefully, form-retaining member 10 canbe constructed from translucent, or transparent material, includingoptical quality transparent material, thus permitting strip 20 to bevisualized while engaged in cavity 18. Typically, form-retaining member10 is constructed of materials that are substantially electricallynonconducting, thus reducing or eliminating the concurrent action onstrip 20 of electrical fields other than those along the channel currentflow axis through cavity 18.

[0126] In typical embodiments, form-retaining member 10 is composed ofceramic, quartz, glass, silicon and its derivatives, plastic, ormixtures thereof. Among plastics useful in the construction ofform-retaining member 10 are polymethylacrylic, polyethylene,polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride,polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal,polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose,polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline,polyester, and mixtures or copolymers thereof.

[0127] Form-retaining member 10 is also usefully composed of materialsthat permit heat to be conducted away from strip 20 duringelectrophoresis. In that regard, form-retaining member 10 can usefullybe shaped to include one or more recessed regions 27, shown in FIGS. 3Cand 3D, reducing the thickness of form-retaining member 10 in regionsproximal to channels 12, reducing thermal resistance between strip 20and a heat sink, usefully a fluid filled chamber, as further discussedbelow.

[0128] Form-retaining member 10 confers dimensional integrity uponchannels 12. Dimensional integrity is important to permit the dispensinginto channel 12 of rehydration solution (optionally with sample to befractionated), to permit strip 20 to be inserted into channel 20, and toeffect hydratable lodging of strip 20 in channel 12 upon rehydration.

[0129] Form-retaining member 10 can confer dimensional integrity uponchannel 12 by contributing at least a portion of the circumferentialwall of cavity 18 of channel 12.

[0130] For example, cavity 18 of channel 12 can be constructed as atunnel, bore, or conduit within form-retaining member 10. In suchembodiments, form-retaining member 10 contributes the entirety of thecircumferential wall of cavity 18.

[0131] Alternatively, cavity 18 can be partially enclosed withinform-retaining member 10, with only a portion of the circumferentialcavity wall of cavity 18 contributed by member 10. In these latterembodiments, channels 12 can be machined into form-retaining member 10,or, depending on the composition of form-retaining member 10,lithographed, engraved, isotropically or anisotropically etched, milled,mechanically or chemically polished, or molded into form-retainingmember 10. Alternatively, in these latter embodiments channels 12 can befabricated on form-retaining member 10 from silicon or resin deposits orslabs.

[0132] In embodiments in which cavities 18 are not fully enclosed byinflexible member 10, channels 12 can be rendered fluidly enclosingalong cavity 18 by physical attachment to form-retaining member 10 ofone or more additional laminae.

[0133]FIG. 4 is an exploded side perspective view of a multilaminateembodiment of cassette 100 of the present invention.

[0134] In the embodiment shown in FIG. 4, form-retaining member 10includes depression 13. Laminate cover 42 includes a plurality ofentries 50. Upon attachment of laminate cover 42 to form-retainingmember 10, depression 13 becomes fluidly enclosing along cavity 18, thuscompleting channel 12, with entries 50 contributing to channel entries14 and 16.

[0135] As with form-retaining member 10, laminate cover 42 can usefullybe optically translucent or transparent, and is usefully substantiallyelectrically insulating.

[0136] As with form-retaining member 10, laminate cover 42 can becomposed of ceramic, quartz, glass, silicon and its derivatives,alumina, polymer, plastic, or mixtures thereof. Among plastics useful inthe construction of laminate cover 42 are polymethylacrylic,polyethylene, polypropylene, polyacrylate, polymethylmethacrylate,polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate,polyacetal, polysulfone, celluloseacetate, cellulosenitrate,nitrocellulose, polystyrene, polyacrylonitrile, polyurethane,polyamides, polyaniline, polyester, and mixtures and copolymers thereof.

[0137] Laminate cover 42 can usefully be, and is often preferably,flexible. Although laminate cover 42 can be of any thickness, to conferflexibility laminate cover 42 can usefully be a film.

[0138] Laminate cover 42 can be attached to form-retaining member 10 bybonding means known in the microfabrication arts, including thermalwelding, ultrasonic welding, and application of adhesives or adhesivelayers.

[0139] For example, U.S. Pat. Nos. 5,800,690 and 5,699,157, incorporatedherein by reference in their entireties, describe methods for completingchannels by attaching planar cover elements to micromachined substratesby thermal bonding, application of adhesives, or by natural adhesionbetween the two components. U.S. Pat. No. 5,593,838, incorporated hereinby reference, teaches that localized application of electric fieldspermits the meltable attachment of a cover element at about 700° C.,well below the flow temperature of silicon (about 1400° C.) or ofCorning 7059 glass (about 844° C.). WO 96/04547 (Lockheed Martin EnergySystems), incorporated herein by reference in its entirety, teaches thata cover plate can be bonded directly to a glass substrate aftertreatment in dilute NH₄OH/H₂O₂, followed by annealing at 500° C., wellbelow the flow temperature of silicon-based substrates. WO 98/45693(Aclara Biosciences), incorporated herein by reference in its entirety,discloses a thermal bonding method for fabricating enclosed microchannelstructures in polymeric, particularly plastic, substrates, an adhesivemethod in which adhesive is applied in a film no more than 2 μm thick,and methods in which fluid curable adhesives are rendered nonflowable bypartial curing before apposition of adherends.

[0140] Laminate cover 42 is usefully attached to form-retaining member10 by reversible bonding means, thus permitting the user to separatelaminate cover 42 from form-retaining member 10 after completion ofelectrophoresis, which in turn permits strip 20 to be removed fromchannel 12 for further processing. Constructing laminate cover 42 as aflexible film offers advantages in such user-mediated separation oflaminate cover 42 from form-retaining member 10.

[0141] In the embodiment depicted in FIG. 4, laminate cover 42 isattached adhesively to form-retaining member 10 using double-sidedlaminate adhesive layer 46.

[0142] As shown, double-sided laminate adhesive layer 46 has elongateslots 48 that are congruent with depressions 13. Such slots 48 preventcontact between double-sided adhesive layer 46 and strip 20 when strip20 is movably inserted into channel 12; contact with adhesive caninterfere with movable insertion of strip 20 into cassette 100.

[0143] In multilaminate embodiments of cassette 100 in which laminatecover 42 is attached with a double-sided adhesive layer 46, thethickness of adhesive layer 46 can be adjusted to change the internaldiameter of cavity 18 of channel 12, thus accommodating hydratable stripmedia of different thicknesses.

[0144] In alternative multilaminate embodiments of cassette 100,laminate cover 42 is itself fashioned as a form-retaining member,typically thicker than the flexible film above-described. In some ofthese embodiments, laminate cover 42 is fashioned as a discretestructure. In other embodiments, form-retaining laminate cover 42 andform-retaining member 10 are movably attached to one other, as by ahinge, or plurality of hinges, present therebetween. The hinge need notitself be fashioned as a separate, intermediating, structure, but caninstead be fashioned as a foldable seam between form-retaining member 10and laminate cover 42. Such seams are common in plastic cases designedto hold, e.g., drill bits.

[0145] In cases in which laminate cover 42 is form-retaining, it can beassembled to form-retaining member 10 by, e.g., snapping laminate cover42 to form-retaining member 10. A pressure compliant surface, onform-retaining member 10 and/or laminate cover 42, facilitates sealingof the two layers, forming an enclosing member suitable forelectrophoresis. Although assembly by snapping of laminate cover 42 toform-retaining member 10 has been described with particularity, anyother mechanical engagement approach, such as mating of tongue andgroove, insertion of a tab into a slot, etc., can also be used tosimilar effect.

[0146] In multilaminate embodiments of cassette 100—both those withflexible and those with form-retaining laminate covers—the internaldiameter of cavities 18 can be adjusted by adjusting the depth ofincursion of channel 12 into form-retaining member 10. In multilaminateembodiments of cassette 100 in which laminate cover 42 is thicker than afilm, the internal diameter of cavities 18 can be adjusted additionallyby adjusting the depth of incursion of channel 12 into laminate cover42.

[0147] Channel 12 is so dimensioned—in both multilaminate and unitaryembodiments of cassette 100—as to permit insertion of a prior-casthydratable strip-based electrophoresis medium, such as an IPG strip, inits dehydrated state, and to lodgingly enclose the strip afterhydration.

[0148] Immobiline DryStrip IPG strips, presently available commerciallyfrom Amersham Biosciences, (Piscataway, N.J., USA), have an approximatewidth of 3 mm and a depth of 0.5 mm. Accordingly, to permitelectrophoresis of these commercial IPG strips, channel 12 of cassette100 will have a width of at least about 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm,3.4 mm, and even 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, andeven 4.1 mm, and will have depth of at least about 0.5 mm, 0.6 mm, 0.61mm, 0.62 mm, 0.63 mm, 0.64 mm, 0.65 mm, 0.66 mm, 0.67 mm, 0.68 mm, 0.69mm, and even 0.7 mm, 0.71 mm, 0.72 mm, 0.73 mm, 0.74 mm, 0.75 mm, 0.76mm, and even 0.77 mm so as to movingly engage such strips in theirdehydrated state and lodgingly enclose the strips when rehydrated.

[0149] ReadyStrip IPG strips, presently available commercially fromBio-Rad (Hercules, Calif., USA) have strip width of 3.3 mm and gelthickness of 0.5 mm. Accordingly, to permit electrophoresis of thesecommercial IPG strips, channel 12 of cassette 100 will have anapproximate width of at least about 3.3 mm, 3.4 mm, and even and even3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, and even 4.1 mm, andwill have depth of at least about 0.5 mm, 0.6 mm, 0.61 mm, 0.62 mm, 0.63mm, 0.64 mm, 0.65 mm, 0.66 mm, 0.67 mm, 0.68 mm, 0.69 mm, and even 0.7mm, so as to movingly engage such strips in their dehydrated state andlodgingly enclose the strips when rehydrated.

[0150] In a presently preferred embodiment, suitable for electrophoresisof strips from both manufacturers, channels 12 have width of 3.7 mm anddepth of 0.64 mm.

[0151] As would be expected, prior-cast hydratable electrophoreticseparation media can, and likely will, be manufactured with dimensionsdifferent from those presently used. Accordingly, cassettes 100 of thepresent invention are not limited to those dimensioned for use with theabove-described strips.

[0152] Design of the internal dimensions of channel 12, so as to permitinsertion of prior-cast hydratable strip based media in their dehydratedstate and lodgingly enclose the strips when rehydrated, is well withinthe skill in the art.

[0153] A simple test for suitability of the internal dimensions ofchannel 12 for a prior-cast hydratable electrophoretic separation medium20 of any given depth and width is as follows:

[0154] (1) Position the cassette horizontally and fill channel 12 withwater;

[0155] (2) Insert strip 20 through entry 14 or through entry 16 intochannel 12 and advance as far as possible by hand;

[0156] (3) After 8 hours, bring cassette 100 to the vertical positionand observe.

[0157] Dimensions of channel 12 are suitable if, in step (2), strip 20can be advanced into channel 12 to a point at which less than 1 cm ofstrip 20 remains outside the entry chosen for insertion, and if, in step(3), air does not directly contact more than 50% of the enclosedseparation medium. Strip 20 should also remain lodged within thecassette once the cassette is brought vertical in step (3).

[0158] At one end of the useable spectrum of channel dimensions, theswelling of the separation medium causes direct, occlusive, contact ofthe separation medium with the channel's internal wall alongsubstantially all of the channel cavity. In this case, a visibly labeledsolution (such as 0.2% w/v bromphenol blue in water) applied to thesuperior channel entry will be substantially precluded from the channelcavity. That is, a visibly labeled solution will typically not extendmore than about 0.25 cm beyond the channel entry into the channelcavity. At the other end of the useable spectrum of channel dimensions,the swelling of the separation medium is insufficient to cause occlusivecontact of the separation medium with the channel's internal wall alongsubstantially all of the channel cavity. In this latter case, a visiblylabeled solution such as 0.2% w/v bromphenol blue in water will enterthe channel cavity from the superior entry when the cassette is broughtvertical. In neither case, however, will air contact more than 50% ofthe enclosed separation medium.

[0159] An additional, functional test for suitability of the internaldimensions of channel 12 for a prior-cast hydratable electrophoreticseparation medium of given dimensions is to replace step (3) of the testset forth above with an actual electrophoresis experiment; dimensions ofchannel 12 are suitable if, in step (2), strip 20 can be advanced intochannel 12 to a point at which less than 1 cm of strip 20 remainsoutside the entry chosen for insertion, and if, after electrophoresis,adequate electrophoretic separation is achieved.

[0160] If strip 20 is an IPG strip, this latter test may usefully beperformed as follows.

[0161] Mix 5.0 μL of Serva IEF standard (catalogue no. 39212-01, ServaElectrophoresis GmbH, Heidelberg, Germany) with sufficient rehydrationbuffer of the following composition to fill the channel: 8.0 M urea,0.5% ampholytes (3-10 IPG buffer, cat. no. 17-6001-11, AmershamBiosciences), 2.0% (w/v) CHAPS, 20 mM DTT, 0.0025% (w/v) bromphenolblue. Pipette the solution into a channel of the cassette with thecassette positioned horizontally. Insert the strip into the channel sothat about 3 mm overextends the channel entries. Occlude the channelentries with cover tape and allow the strip to rehydrate for 8-16 hours.Remove cover tape and, if present, loading wells.

[0162] Apply an electrode wick (further described below) to each set ofentries. Evenly apply 750 μl of deionized water to each electrode wick.

[0163] Contact the cassette to the electrodes of a buffer core (furtherdescribed hereinbelow). Apply a buffer dam (further described below) tothe other contact face of the buffer core. Slide the buffer core into anelectrophoresis chamber and fill the outer chamber surrounding thebuffer core with water. Take care that the water does not overtop thecassette and spill into the inner chamber (the outer walls of which aredefined by the cassettes and buffer core).

[0164] Apply a voltage in three steps according to the followingprofile: 200 V for 20 min, 450 V for 15 minutes, 750 V for 15 minutes,2000 volts for 30 minutes.

[0165] Channel dimensions are suitable if discrete marker bands areobservable.

[0166] Channel entries 14 and 16 will typically, but not invariably, bespaced so that channel 12 engages substantially the entire length ofstrip 20, as shown e.g. in FIG. 2.

[0167] IPG strips are currently available commercially in a variety oflengths. For example, Immobiline DryStrip IPG strips, presentlycommercially available from Amersham Biosciences, (Piscataway, N.J.,USA), are available with gel lengths of 70 mm, 110 mm, 130 mm, 180 mm,and 240 mm. ReadyStrip IPG strips, presently commercially available fromBio-Rad (Hercules, Calif., USA), are available with gel lengths of 70mm, 110 and 170 mm. ZOOM® IPG strips presently commercially availablefrom Invitrogen (Carlsbad, Calif., USA) have gel lengths of 70 mm.

[0168] Thus, in certain presently preferred embodiments of cassette 100of the present invention, channels 12 are fashioned to accommodatesubstantially the entire length of strips with gel lengths of 70 mm, 110mm, 170 mm, 180 mm, and 240 mm in length.

[0169] In such commercial IPG strips, the polyester backing typicallyextends for some distance beyond the gel on either end. Thus, channels12 will typically have length at least as long as the stated gel length(70, 100, 170, 180, or 240 mm), typically with extension of 1 mm, 2 mm,3 mm, 4 mm, 5 mm, or even 6 mm on both ends. Thus, for an IPG strip ofnominal 70 mm gel length, channel 12 will be at least about 70 mm inlength, 72 mm in length, 74 mm in length, 76 mm in length, 78 mm inlength, and even 80 or 82 mm in length. In a presently preferredembodiment for IPG strips of 70 mm stated gel length, channel 12 will be80 mm in length.

[0170] It would be expected that rehydratable strip-based separationmedia will in the future be available in a variety of lengths, just asthey are expected to be available in a variety of widths and depths, asdescribed above. It is, therefore, an aspect of the invention to providecassettes 100 with channels 12 dimensioned to engage prior-casthydratable electrophoretic separation media of any chosen length.

[0171] As suggested above, significant overextension or underextensionof channel 12 by strip 20 is undesirable.

[0172] For example, if strip 20 extends substantially beyond entry 14,entry 16, or both, the overextending portion(s) of strip 20 will beexposed to atmospheric CO₂, obviating an important advantage of thepresent invention. Furthermore, the overextending portion(s) of strip 20can permit leakage of ampholyte and/or protein from the strip.Additionally, only that portion of the separation medium lying betweenthe spaced electrical connections will be functionally available forseparation, reducing the functional portion of gel. Finally, theoverextending portion(s) might interfere mechanically with establishmentof electrical communication properly required for electrophoresis. Andwhen strip 20 underextends channel 12, it can prove difficult toestablish effective electrical communication with the enclosed strip.

[0173] To accommodate these difficulties in a cassette having channelsof nonoptimal length, if strip 20 overextends channel 12, excess can beremoved using scissors or knife; typically, only that portion of strip20 lacking separation media will be so removed. If strip 20 underextendschannel 12, the recessed end can be brought into effective electricalcommunication with the exterior of channel 12 by filling the recessedend with an electrically conductive, channel-filling, material.

[0174] Among materials usefully employed to bring the underextended endof strip 20 into electrical communication with an entry 14 or 16 ofcassette 100 are materials that can be applied in liquid or semiliquidstate, in which state they can conform in shape to the channel interior,and that thereafter polymerize or gel into a shape-holding phase.

[0175] Usefully, the material can be a polymer gel, such as agarose.When so used, the agarose can be rendered molten in the presence ofelectrolyte-containing buffer, such as rehydration solution, applied toentry 14, entry 16, or both as a molten liquid, and thereafter allowedspontaneously to gel with decrease in temperature. Polyacrylamide canalso be used, although in this latter case polymerization of monomersand cross-linkers must be effected by addition of catalyst, as is wellknown in the art.

[0176] Usefully, cassette 100 includes a plurality of channels 12. Incases in which cassette 100 includes a plurality of channels 12, thecurrent flow axes of plural channels 12 are usefully substantiallyparallel to one another, and cavities 18 of plural channels 12 arefluidly noncommunicating with one another except at channel entries 14and 16.

[0177] In such embodiments, channels 12 need not have identical cavity18 dimensions, a single cassette 100 thus accommodating strips 20 ofdifferent dimensions. Typically, however, cavities 18 of plural channels12 will all have the same internal dimensions.

[0178] Although cassette 100 is described above as permittinguser-directed insertion of strip 20 into channel 12, it is anotheraspect of the present invention to provide a cassette, asabove-described, in which strips 20 have already been inserted duringmanufacture. Such cassettes 100 can usefully be disposable.

[0179] To facilitate sample application, and in particular to facilitatesample application without cross contamination as among plural channels12, cassette 100 can usefully include loading wells. FIG. 1A shows oneembodiment of such loading wells; FIGS. 1B and 1C show anotherembodiment of such loading wells.

[0180] With reference to FIG. 1A, cassette 100 is shown to have twowell-forming members 22. The two well-forming members define discretereservoirs, termed loading wells, at each of the six entries 14 and sixentries 16, respectively. When cassette 100 is horizontal withwell-forming members 22 superior to form-retaining member 10, eachloading well can maintain a defined maximum volume of fluid in contactwith an entry 14 (or entry 16) without cross-over fluid contact withadjacent entries.

[0181] In cases in which sample to be fractionated is applied afterinsertion of strip 20, the loading wells permit samples of volume lessthan the maximum reservoir volume to be applied discretely to individualwells 14 (and/or 16) without cross-over contamination. In cases in whichsample is applied in rehydration buffer prior to insertion of strips 20into channels 12, the loading wells prevent cross-over contamination bysample displaced from channel 12 during strip insertion.

[0182] After sample to be fractionated (such as a protein sample forisoelectric focusing on IPG strips) enters the separation medium ofstrip 20, cross-over contamination among channels 12 is usuallyforeclosed, even if entries 14 are thereafter placed in fluidcommunication with one another and entries 16 are thereafter placed influid communication with one another. Accordingly, well-forming members22 can be removable. Such removal can facilitate subsequent applicationof conductive wicks 24, as shown in FIG. 3B and further described below.

[0183] Because well-forming member 22 is typically removed prior toelectrophoresis, there are fewer constraints on the materials from whichit can be constructed than for form-retaining member 10 and, inmultilaminate embodiments of cassette 100, for laminate cover 42.Indeed, well-forming member 22 can be constructed of any material thatis substantially chemically unreactive with the rehydration solution,such as ceramic, quartz, glass, silicon and its derivatives, plastic,natural or synthetic rubber polymers, or mixtures thereof. Amongplastics useful in the construction of well-forming member 22 arepolymethylacrylic, polyethylene, polypropylene, polyacrylate,polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene,polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate,cellulosenitrate, nitrocellulose, polystyrene, polyacrylonitrile,polyurethane, polyamides, polyaniline, and mixtures thereof. Siliconeand its derivatives are also useful.

[0184] In certain embodiments, well-forming member 22 can be composed ofelectrically conductive materials; this facilitates “active rehydration”of strip 20. In “active rehydration”, strip 20 is rehydrated in thepresence of a low voltage gradient, approximately 100 V, establishedalong the channel current flow axis of strip 20 between entries 14 and16.

[0185] In cases in which active rehydration is desired, well-formingmember 22 can be composed of an electrically-conductive material, suchas an electrically-conductive polymer, such as a polymer impregnated ordoped with carbon. After both strip 20 and rehydration solution areapplied to channel 12 (in either order), a cathode is contacted to firstconductive well-forming member 22 and an anode is contacted to secondconductive well-forming member 22 and a voltage applied during therehydration period. The anode and cathode can be, e.g., an electrodebar, such as is found on the MultiPhor (Amersham Biosciences,Piscataway, N.J.) or Blue Horizon (Serva, Heidelberg, Germany) devices.

[0186] When cassette 100 is unitary—that is, having channels 12 formedcompletely within form-retaining member 10—well-forming members 22 canbe attached to form-retaining member 10. When cassette 100 is, instead,multilaminate—e.g., with channels 12 formed in part by a laminate cover42—well-forming members 22 can be attached to laminate cover 42, asshown in FIG. 5.

[0187]FIG. 5 is an exploded side perspective view showing well-formingmembers 22 attached adhesively to laminate cover 42 using double-sidedwell-forming member adhesive layer 54. However, as described above withrespect to attachment of laminate cover 42 to form-retaining member 10,which discussion is incorporated herein by reference, well-formingmember 22 can be attached to laminate cover 42 by a variety of bondingmeans well known in the microfabrication arts, including thermalwelding, ultrasonic welding, and application of liquid or partiallycured adhesives, as well as by means of adhesive layers.

[0188] Well-forming member 22 can in the alternative be attached tolaminate cover 42 by engagement of opposing, matching surfaces, as in asnap, or engagement of tongue with groove, or engagement of tab withslot.

[0189] However bonded, well forming members 22 will usefully bereversibly attached to cassette 100, thus permitting removal of thewell-forming members prior to electrophoresis. In cases in whichattachment is by means of a double-sided well-forming member adhesivelayer 54, the adhesive layer is usefully designed to adhere morestrongly to form-retaining member 10 (or, in multilaminate embodiments,to laminate cover 42) than to well-forming member 22; in such adhesivelybiased embodiments, removal of well-forming member 22 will typicallyleave adhesive layer 54 on form-retaining member 10 (or laminate cover42), facilitating application of conductive wicks, as further describedbelow.

[0190]FIG. 3A shows an embodiment of the cassette of the presentinvention with well-forming members 22 removed, prior to application ofconductive wicks, as further described below.

[0191] Although cross-contamination of samples as among plural channels12 will typically be foreclosed by entry of sample into the separationmedium of strip 20, thus obviating the requirement for continuedpresence of well-forming members 22 during electrophoresis, it cannonetheless be advantageous further to seal entries 14 and/or 16 aftersample application.

[0192] In these latter embodiments, sealing is accomplished byapplication to entries 14 and/or 16 of a material that is electricallyconductive, that can be applied in a state in which it conforms in shapeto the entry and/or loading well, and that thereafter polymerizes orgels into a shape-holding phase. As above, such material can usefully bea polymer gel, such as agarose or acrylamide.

[0193] In particularly useful approaches, entries 14 and/or 16 aresealed with an amount of material sufficient to fill channel 12 andentry 14 (and/or entry 16) to a level flush with the surface ofform-retaining member 10. Such geometry facilitates electrical contactof the anodic and cathodic ends of strip 20 directly or indirectly withanode and cathode electrodes.

[0194] Returning to FIG. 1A, cassette 100 can optionally, and usefully,include ribs 40.

[0195] Ribs 40 facilitate alignment of laminate cover 42 andwell-forming members 22 during manufacture of cassette 100. Ribs 40 canalso facilitate proper operational engagement of cassette 100 by anelectrophoresis chamber or buffer core, as further described below.

[0196] Ribs 40 can be machined or molded directly from form-retainingmember 10, or can be separately constructed and fixed thereto. Whenseparately constructed, ribs 40 are usefully constructed of solid orsemisolid materials that are readily machined, molded, or etched, andthat are chemically compatible—that is, do not suffer substantialdegradation upon contact—with electrophoretic buffer systems. Usefully,ribs 40 can be constructed of materials that are substantiallyelectrically insulating, including ceramic, quartz, glass, silicon andits derivatives, or plastic, or mixtures thereof. Among plastics usefulin the construction of ribs 40 are polymethylacrylic, polyethylene,polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride,polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal,polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose,polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline,polyester, and mixtures and copolymers thereof.

[0197] As noted above, after rehydration of and introduction of sampleinto strip 20, strip 20 becomes lodgingly enclosed in cavity 18 ofchannel 12. With strip 20 so enclosed, electrophoresis can then beperformed, without removing strip 20 from cassette 100, by applying avoltage gradient to flow current through strip 20 along the channelcurrent flow axis sufficient to effect electrophoretic separation ofanalytes therein.

[0198]FIG. 3B illustrates one useful, but nonlimiting, approach by whichcassette-enclosed strip 20 is rendered contactable by cathode and anodeelectrodes to complete the necessary electrical circuit.

[0199]FIG. 3B is a front perspective view of a cassette of the presentinvention having six channels 12. As shown, a first conductive wick 24contacts strips 20 (present in three of six available channels 12) atentries 14; a second conductive wick 24 contacts strips 20 at entries16.

[0200] Wick 24 includes an electrically conductive material. Thematerial need not be constitutively conductive: it suffices, and indeedtypically will be the case, that wick 24 is conductive when wet. In thislatter case, wick 24 can usefully be composed of a bibulous material,such as paper, nitrocellulose, felt, nylon, or derivatives thereof.

[0201] As described above, as an alternative or in addition to thepresence of wicks 24, strip 20 can be electrically coupled to cathodeand anode electrodes through intermediation of electrically conductivepolymers or hydrogels such as agarose.

[0202] As shown in FIG. 3B, first conductive wick 24 can usefullycontact each of plural entries 14, and second conductive wick 24 canusefully contact each of plural entries 16, facilitating application ofcurrent in parallel to plural channels 12. While useful, such geometryis not required.

[0203] First conductive wick 24 is then contacted with an electrode,serving as either cathode or anode. The choice as between applying acathode or anode to wick 24 depends upon the intended electrophoretictechnique, the location of sample application, and other conditions wellknown to those in the electrophoretic arts. For example, for isoelectricfocusing using IPG strips, where one end of the strip is acidic and theother basic, the basic end of the strip is preferably placed inelectrical communication with the cathodic electrode.

[0204] Second conductive wick 24 is then contacted with an electrode (ananode if first wick 24 is contacted with the cathode, a cathode if firstwick 24 is contacted with the anode).

[0205] Any means of electrode attachment to wicks 24 can be used, aslong as effective electrical communication is established.

[0206] In an alternative to use of conductive wicks 24, spacedelectrical communication with enclosed strip 20 can be effected bydirect contact of strip 20 with anode and cathode electrodes. Contactcan be accomplished by passage of anode and cathode electrodes throughentries 14 or 16, or alternatively by passage of electrodes throughform-retaining member 10 or laminate cover 42 elsewhere than at entries14 and/or 16. As an example of the latter approach, electrodes shaped asblades can be used to pierce laminate cover 42 in embodiments in whichlaminate cover 42 is a flexible film, thereby contacting enclosed strip20 at spaced intervals.

[0207] Electrophoresis can thereafter be conducted with cassette 100 inany physical orientation. In a particularly useful approach, electrodecontact is effected using an adaptor that permits electrophoresis to beconducted with cassette 100 maintained vertically; even when cassette100 is held vertical, channels 12 of cassette 100 can be horizontal orvertical, as desired.

[0208] Returning to FIG. 3B, it is, therefore, another aspect of theinvention to provide an adaptor that permits cassettes 100 of thepresent invention, within which are lodgingly enclosed strips 20, to beelectrophoresed in a vertical direction. It should be noted that evenwhen cassette 100 is itself oriented vertically, channels 12 can stillbe oriented horizontally; in such an orientation, channels 12 ofcassette 100, if present plurally, would be spaced with vertical offsetfrom one another. For clarity, therefore, the term “vertical” isintended to refer to the orientation of the cassette, not the channels.

[0209] Electrophoresis of cassette 100 in the vertical dimension has thesignificant advantage of reducing the bench footprint of theelectrophoresis device, freeing up valuable bench space for otherequipment or uses.

[0210] Furthermore, modular electrophoresis systems for performing slabgel electrophoresis in the vertical dimension are well known, see e.g.U.S. Pat. Nos. 5,888,369 and 6,001,233, and are commercially available(Invitrogen, Carlsbad, Calif., USA; Bio-Rad, Hercules, Calif., USA). Inpreferred embodiments, the adaptor of the present invention permitscassettes 100 of the present invention to be electrophoresed in suchexisting modular electrophoresis systems, permitting the efficient useof such prior-purchased equipment for electrophoresis of prior-casthydratable electrophoretic separation media, such as IPG strips.

[0211]FIG. 6A is a front perspective view of an adaptor, herein termed abuffer core, of the present invention. FIG. 6B is a front perspectiveview of the buffer core (front) operationally aligned with, but not yetcontacting, a cassette of the present invention (rear); operationalcontact is shown in FIG. 6C. As can be seen, buffer core 26 is designedsimultaneously to align cathode electrode wire 31 with cathodic wick 24of cassette 100 and anode electrode wire 32 with anodic wick 24 ofcassette 100.

[0212] As shown in FIG. 6B, cathode wire 31 is attached at a first endto cathode contact prong 38; analogously, anode wire 32 is attached at afirst end to anode contact prong 36. Contact prongs 38 and 36 permit theremovable attachment of wires having standard female gender plugs; as iswell known in the electrophoresis arts, the other end of such wires istypically connected to a power supply, such as a regulatable powersupply.

[0213] Also as shown, cathode wire 31 extends from cathode contact prong38 to-support 28 before terminating at a second end, and anode wire 32extends from anode contact-prong 36 to support 30 before terminating ata second end. Supports 28 and 30 are typically composed of materialsthat are substantially electrically insulating and substantially inertto electrophoresis running buffers. For example, supports 28 and 30 areconveniently made of plastic, such as polycarbonate.

[0214] Contact between anode wire 32, cathode wire 31, and theirrespective wicks 24 of cassette 100 is effected by application of anexternal compressive force applied inwardly against cassette 100. Theanode and cathode wires are thereby compressed between their respectivesupport and wick, establishing an electrical connection.

[0215] As shown in FIG. 6D, buffer core 26 can, and typically will, beoperationally aligned and contacted simultaneously with a secondcassette 100. So aligned and so contacted, buffer core 26 and cassettes100 define an internal chamber 62, open only at the top and sealed,except from above, from external liquids. If the number of strips neededto be electrophoresed can be accommodated in a single cassette, a“buffer dam”, dimensioned similarly to cassette 100 but lacking channels12 can be used to complete buffer core internal chamber 62.

[0216] In order to conduct electrophoresis using cassettes and buffercores of the present invention, cassettes 100 (or singular cassette 100and a buffer dam) are aligned and contacted to buffer core 26. Theassembly is then engaged in electrophoresis buffer chamber 34 whichitself, or in conjunction with an additional device, urges cassettes 100(or singular cassette 100 and buffer dam) into sealable contact withbuffer core 26. Such additional urging device can be a cam-activatedclamp (“tension wedge”), as further described in U.S. Pat. No.6,001,233, incorporated herein by reference in its entirety.

[0217] Alternatively, buffer core 26 is first loosely engaged inelectrophoresis buffer chamber 34, and cassettes thereafter aligned,contacted to, and then further urged against buffer core 26.

[0218] Fluid-tight contact between buffer core 26 and cassettes 100 (orsingular cassette 100 and buffer dam) is typically, but optionally,further facilitated by a gasket, such as a silicone gasket, fitted intogroove 70 of buffer core 26, shown in FIGS. 6A and 6D.

[0219] As noted above, buffer core 26 and cassettes 100 (or singularcassette 100 and a buffer dam) in sealed engagement therewith defineinternal chamber 62. This chamber isolates cathode wire 31 and anodewire 32 from fluids present external to buffer core 26 inelectrophoresis chamber 34 (chamber 60), so long as the fluid level inelectrophoresis chamber 60 does not over top cassettes 100.

[0220] Accordingly, electrophoresis chamber 34 can be filled with anychosen liquid solution, to a level that does not overtop cassettes 100,without affecting the electrical circuit. Such fluids can thus usefullyserve as a heat sink, reducing the temperature of strips 20 as they aresubjected to current flow in cassettes 100.

[0221] Electrophoresis is conducted by attaching, via contact prongs 36and 38, anode and cathode to a power supply. Conveniently, this may beperformed by applying a cover having integrated electrodes, asillustrated in FIG. 11. The cover may usefully be designed so as to fitexisting electrophoresis chambers, permitting their use with the presentinvention, but to interface only with buffer cores of the presentinvention.

[0222] A potential difference (voltage gradient) is applied that issufficient to effect separation of analytes within the separation mediumof strip 20. In embodiments in which strip 20 is an IPG strip, proteins,influenced by the voltage gradient, begin to migrate until the pI of theprotein coincides with the pH on the immobilized gradient, at whichpoint the focused protein ceases to move.

[0223] The voltage difference actually achieved by spaced electricalcontact with strip 20 will always be less than that reported by thepower supply, due to resistances inherent in the various connectionsbetween power supply and gel. In the cassette and buffer core of thepresent invention, such parasitic impedances include, e.g., (i) cableconnections to the power supply, (ii) resistances in the cablesthemselves, (iii) cable-to-electrode contact resistance, (iv) electrodeto wick contact resistance, (v) impedance of the wick itself, which is afunction of the ionic concentration in the moist wick, which can varyduring the run due to either endosmosis or diffusion, (vi) contactresistance between the wick and gel surface, which is influenced by thepressure of that interface, (vii) the inherent impedance of the gel.

[0224] In another aspect, the invention provides apparatus that providesa resistance pathway between power supply and prior-cast hydratableseparation medium that is substantially lower than that found in priorart apparatus used for electrophoresis in the prior art; the reducedresistance substantially increases the efficiency with which prior-casthydratable separation media, such as IPG strips, may be electrophoresed.

[0225] Of the parasitic impedances, the electrode-to-wick andwick-to-gel contact resistances can contribute significantly to theoverall voltage drop from power supply to gel. These contact resistancesdepend upon the respective contact pressures. At any given compressiveforce applied inwardly against cassettes 100, the magnitude of theseelectrically effective contact pressures will depend upon the proportionof the force brought to bear at these locations. Accordingly, supports28 and 30 may usefully be designed to distribute the externalcompressive force discontinuously, creating greater contact pressure atthe first and second channel entries than elsewhere on cassette 100.

[0226] In a first series of exemplary embodiments, illustrated in FIGS.6A -6C, the cassette contact faces of support 28 and support 30 arenonplanar: a plurality of discontinuous indentations collectively definea series of intervening protuberances. The serrated surface so createdis capable of discontinuously distributing external compressive force toanode wire 31 and cathode wire 32.

[0227] In the embodiments shown, the indentations are sufficiently deepas to cause periodic discontinuities in the contact of the electrodewires to their respective supports. Such depth of indentation is notrequired.

[0228] Indentations are positioned so as to align each interveningprotuberance with an entry 14 (equivalently for the other electrode,entry 16) of cassette 100. Indentations are sized so as to createprotuberances with dimensions closely approximated to the lateraldimensions of entries 14 and 16.

[0229] For example, to minimize electrically ineffective contact (andthus maximize the proportion of electrically effective contact) withembodiments of cassette 100 that are capable of holding six strips 20,supports 28 and 30 have (on at least one cassette-contact face) sixprotuberances, each positioned to align with one of the six entries 14(equivalently, entries 16) of cassette 100, and preferably sized to asto approximate the lateral dimensions of entries 14 and 16.

[0230] The faces of support 28 and support 30 that contact the samecassette 100 typically will have the same number of protuberances.However, for each of supports 28 and 30, the two cassette contact facesneed not have the same number of protuberances (as is the case in theembodiments illustrated in FIGS. 6A-6C), if two cassettes 100accommodating different numbers of strips 20 are to be applied to thetwo cassette contact faces. Typically, however, the two cassette contactfaces will have the same number of protuberances.

[0231] In another series of embodiments (not shown), support 28 andsupport 30 lack indentations. Instead, the supports are nonunitary inconstruction, comprising materials that are differentially compressible.The least compressible materials are positioned to align with entries 14(equivalently 16) of cassette 100; the more compressible materials arepositioned to align elsewhere on cassette 100. The different degrees ofcompressibility cause differential distribution of the externalcompressive force, causing increased contact pressure at entries 14 (and16).

[0232] The discontinuously applied pressures—occasioned, for example, byserrating the outward surfaces of supports 28 and 30, as in FIGS.6A-6C—substantially improve the efficiency with which voltage (andcurrent) can be applied to strips 20. Efficiency of voltage (andcurrent) application to IPG strips, defined herein as the ratio, for agiven voltage output from a power supply, of currents measured atidentical strips 20 having identical samples, may be at least 2-foldbetter, 3-fold better, 4-fold better, even at least 5-fold better usingthe cassette and buffer core of the present invention as compared tohorizontal, oil immersion IPG electrophoresis devices of the prior art.Efficiencies may even be at least 6-fold, 7-fold, 8-fold, 9-fold, andeven at least 10-fold better. The data set forth in Examples 2 and 3herein, plotted in FIGS. 9 and 10, demonstrate a 2-4 fold betterefficiency than is observed using a prior art oil immersion flatbed IPGdevice.

[0233] The increased efficiency of electrical transmission providessignificant advantages.

[0234] The increased efficiency permits IPG IEF to be performed usingsubstantially lower power supply voltages and power supply currents,permitting less expensive power supplies, lacking current limitationmeans, to be used. The data set forth in Example 3 below demonstratesthat even simple unregulated power supplies capable only of step voltageprofiles may be used, obviating the need for power supplies capable oframped voltage profiles.

[0235] The increased efficiency permits shorter run times to achieve thevolt-hours required for focusing.

[0236] Using the cassette and buffer core of the present invention,focusing can be achieved in as few as 6 hours, 5 hours, 4 hours, even asfew as 3.5 hours, 3.0 hours, 2.5 hours, 2.0 hours, or even as few as 1.5or 1.25 hours, depending upon the sample, strip length, strip pH range,and voltage profile. Focusing can thus typically be achieved in 1.25-10hours, 1.5-9 hours, 1.75-8 hours, 2-7 hours, 2.5-6 hours, and even in1-3 hours.

[0237] Focusing can thus be achieved at least two times faster than withexisting horizontal flatbed oil-immersion devices, often at least3-times, 4-times, even 5-times faster. At times, focusing can beachieved at least 6-times, 7-times, 8-times, 9-times, even as much as10-times faster.

[0238] For example, using a ramping power supply, a 7 cm IPG gel, acassette capable of holding 6 such strips, and a buffer core withserrated supports, focusing can be achieved using the following rampedvoltage profile:

[0239] 0-175 volts over 15 minutes

[0240] 175-2000 volts over 45 minutes

[0241] 2000 volts for 20-30 minutes.

[0242] Using a stepped power supply, a 7 cm IPG gel, a cassette capableof holding 6 such strips, and a buffer core with serrated supports,focusing can be achieved using the following stepped voltage profile:

[0243] 200 V for 20 minutes

[0244] 450 V for 15 minutes

[0245] 750 V for 15 minutes

[0246] 2000 V for 30 minutes.

[0247] Successful focusing can even be achieved using a constant powersupply, such as the PowerEase® 500 (Invitrogen Corp., Carlsbad, Calif.),by applying

[0248] 500 V for 3-4 hours

[0249] to a 7 cm IPG strip.

[0250] Accordingly, using the cassette and buffer core of the presentinvention, IPG isoelectric focusing can be achieved using maximalvoltages as low as 3500 V, 3000 V, 2750 V, 2500 V, 2250 V, 2000 V, 1750V, 1500 V, 1250V, 1000 V, and even as low as 750 V or 500 V, and may beachieved using a ramped voltage profile, stepped voltage profile, or aconstant voltage profile. Minimal voltages may be 500 V, 750 V, 1000 V,1250 V, 1500 V, 1750 V, 2000 V, 2250 V, even 2500 V or more.

[0251] Typically, electrical parameters required for effectiveisoelectric focusing using IPG strips are recited as a minimum, ordesired, number of volt-hours, where volt-hours are defined as thecumulative sum of voltage times hours for each stage of a profile. Asnoted above, the volts nominally reported by the power supply are alwaysgreater than the voltages actually applied across the strip, the ratioof nominal to actual volt-hours depending upon the efficiency of theapparatus.

[0252] Prior art approaches to IEF using IPG strips typically call forat least 13,000 nominal volt-hours; some separations require as many as36,000 nominal volt-hours.

[0253] The increased efficiency of the cassette and buffer core of thepresent invention permit focusing to be achieved in fewer than 13,000nominal volt-hours, typically in fewer than 12,000 nominal volt-hours,11,000 volt hours, 10,000 nominal volt hours, 9000 nominal volt hours,8000 nominal volt hours, 7000 nominal volt-hours, 6000 nominalvolt-hours, 5000 nominal volt-hours, 4000 nominal volt-hours, 3000nominal volt-hours, even as few as 2000, 1900, 1800, 1700, 1600, 1500,1400 or as few as 1300, 1200, 1100, or 1000 nominal volt-hours, forstrips of 70 mm, 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 120 mm, 130 mm,140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, 200 mm, 210 mm, 220 mm,240 mm, or 240 mm, and for shorter, longer, or intermediate lengths.

[0254] Thus, focusing may be achieved after focusing for 1000-12,000nominal volt-hours, 1100-11,000 nominal volt-hours, 1200-10,000 nominalvolt-hours, 1300-9000 nominal volt-hours, 1400-8000 nominal volt-hours,and 1500-7000 nominal volt-hours, for strips of 70 mm, 80 mm, 90 mm, 100mm, 110 mm, 120 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180mm, 190 mm, 200 mm, 210 mm, 220 mm, 240 mm, or 240 mm, and for shorter,longer, and intermediate length gels.

[0255] As is well known in the art, optimal voltage profiles and timeswill vary depending upon the sample, and may vary depending upon thepower supply, the number of strips focused concurrently, and othervariables known in the art, such as the pH range of the IPG strip. Forexample, pH 6-10 IPG strips may need to be focused for 30 minuteslonger; similarly, crude protein mixtures or samples containing highsalt concentration (e.g., >10 mM) may require longer run times or totalvolt hours for optimal resolution. It is well within the skill in theart to optimize focusing conditions empirically by routineexperimentation, such as by varying any one or more of voltage, time,and profiles, starting with the above guidelines.

[0256] The increased electrical efficiency of the cassette and buffercore of the present invention, by permitting lower voltages and moreefficient current application, reduces the propensity of IPG strips toundergo arcing and burning.

[0257] Upon completion of electrophoresis, buffer core 26 with cassettes100 can be stored in a sealed container at −80° C. until strips areready for analysis.

[0258] Strips 20 can, and typically will, be withdrawn from cavity 18for further processing, with or without prior freezing. As describedearlier, although strips 20 can at times be removed upon drying viachannel entries 14 and 16, strips 20 will typically be removed byexpanding the dimensions of cavity 18 of channel 12; in multilaminateembodiments of cassette 100, this is accomplished by separating laminatecover 42 from form-retaining member 10.

[0259] The buffer core embodiment above-described is designed tofacilitate electrophoresis of a cassette in which, for each channelpresent therein, channel entries 14 and 16 permit electricalcommunication with the channel cavity 18 therebetween through a commonsurface of cassette 100, as is shown, e.g., in FIGS. 1-3.

[0260] Such a geometry is not required, however. The invention thusfurther provides a cassette in which entries 14 and 16 do not openthrough the same surface of form-retaining member 10, and a buffer coresuited to electrophoresis of such a cassette. A principal advantage ofsuch a geometry is that it can render the cassette compatible withbuffer cores presently sold for slab gel SDS-polyacrylamide gelelectrophoresis.

[0261]FIG. 7A is a front view, and FIG. 7B a side view, of a cassette1000 of the present invention in which entries 114 and 116 of channels112 respectively open through opposite surfaces of cassette 1000.

[0262] Channels 112 of cassette 1000, like channels 12 of cassette 100,are so dimensioned as to movingly engage a prior-cast hydratableelectrophoretic separation medium in its dehydrated state and lodginglyenclose the strip after rehydration.

[0263] As should be apparent, in order to conduct electrophoresis usingcassette 1000 as the enclosing member, cathode and anode must establishelectrical communication with strip 20 from opposite sides of cassette1000.

[0264] As when entries 14 and 16 open channel 12 to the same face ofcassette 100, so too electrical communication of channel 112 throughentries 114 and 116 can be direct, as by through-passage of electrodesthrough respective entries, or indirect, as by intermediation by polymergels and/or conductive wicks. Additionally, however, when entries 114and 116 open on opposite sides of cassette 1000, electricalcommunication can be established by contact of anode and cathodeelectrodes separately to a first and a second buffer reservoir, whichreservoirs in turn separately contact entries 114 and 116.

[0265] In the latter case, first and second buffer reservoirs must bemaintained in electrical isolation from one another, except by way of acircuit to be completed through the separation medium of strip 20.

[0266] Such geometry can readily be effected by sealingly contactingcassettes 1000, or a singular cassette 1000 and a buffer dam, to abuffer core 126, as further described in commonly-owned U.S. Pat. No.5,888,369, incorporated herein by reference in its entirety, and asavailable commercially from Invitrogen Corp. (XCell II™ Buffer Core withElectrodes, catalogue no. EI9014X, Invitrogen Corp., Carlsbad, Calif.).

[0267] In order to conduct electrophoresis using such system, twocassettes 1000 (or a single cassette 1000 and a buffer dam) arelodgingly engaged in operational alignment with buffer core 126, asshown in FIGS. 7C and 7D.

[0268] The assembled buffer core and cassettes is then engaged inelectrophoresis buffer chamber 34 which itself, or in conjunction withan additional device, urges cassettes 1000 (or singular cassette 1000and buffer dam) into sealable contact with buffer core 126. Suchadditional device can usefully be a cam-activated clamp, such as thatfurther described in U.S. Pat. No. 6,001,233, incorporated herein byreference in its entirety. Alternatively, buffer core 126 is firstloosely engaged in electrophoresis buffer chamber 34, and cassettesthereafter aligned, contacted to, and then further urged against buffercore 126.

[0269] Fluid-tight contact between buffer core 126 and cassette 1000 (ora buffer dam) is typically, but optionally, further facilitated by agasket, such as a silicone gasket, fitted into groove 170 of buffer core126.

[0270] Buffer core 126 and cassettes 1000 (or singular cassette 1000 andbuffer dam) in sealed engagement therewith define internal chamber 162which, if cassettes 1000 are not overtopped, is fluidly noncommunicatingwith electrophoresis buffer chamber 34. A conductive solution is thenadded to internal chamber 162 to a level that (i) contacts cassetteentries 114 (or 116, as the case may be) that open into chamber 162, and(ii) that does not overtop cassettes 1000. A conductive solution is alsoadded to electrophoresis buffer chamber 34 to a level that (i) contactsthe cassette entries 116 (or 114, as the case may be) that open intochamber 34, and (ii) that does not overtop cassettes 1000.

[0271] As further described in commonly-owned U.S. Pat. No. 5,888,369,and well known to users of the XCell™ SureLock system, the electrodegeometry of buffer core 126 effects contact of the anode to internalchamber 126 and cathode to an external reservoir 60 formed in chamber34, thus permitting the requisite voltage gradient to be applied acrossstrip 20 to effect electrophoresis.

[0272] It should be noted that a potential disadvantage of directcontact of channels 112 and strips 20 with liquid reservoirs is theincreased likelihood of ampholyte and/or sample leakage from theseparation medium.

[0273] Although the cassettes of the present invention have beenparticularly described herein above as having at least one prior-formedchannel with sufficient dimensional integrity as to permit the lodgingby hydration of prior-cast hydratable separation media engaged therewithin, prior-formed channels are only one approach to hydratinglylodging such media within an enclosing member.

[0274] By way of example only, the enclosing member, if malleable yetshape-retaining, can be wrapped around the strip in its dehydrated form,fashioning a de novo channel which, upon hydration of the strip,lodgingly encloses the rehydrated strip there within.

[0275] In a further aspect, the present invention provides kits thatfacilitate the practice of the methods of the present invention.

[0276] The kits of the present invention may consist of at least onecomponent (singularly or as a plurality thereof) selected from the groupconsisting of: a buffer core of the present invention, anelectrophoresis chamber, an electrophoresis chamber lid that canestablish electrical communication with a buffer core, a tension wedge,a buffer dam, an electrode wick, sealing tape, an IPG strip,containerized carrier ampholytes, containerized cathode buffer,containerized anode buffer, containerized stains (such as silver stainsor colloidal blue stains), analyte standards, such as protein standards,typically in admixture, and a polyacrylamide gel suitable for performinga second dimension of separation.

[0277] Accordingly, a first series of kit embodiments may includenondisposable items useful for adapting electrophoresis devices toaccommodate cassettes of the present invention. For example, a kit mayinclude a buffer core of the present invention and corresponding lid.

[0278] For users who have neither electrophoresis chamber nor tensionwedge, such as a cam-activated tension wedge, suitable to apply pressurethat urges cassettes 100 inwardly against the buffer core, a kit of thepresent invention may include an electrophoresis chamber, tension wedge,buffer core and lid. Usefully, to permit a single cassette to be run,the kit may additionally optionally comprise a buffer dam.

[0279] A second series of kit embodiments may include disposable itemssuitable for use in the invention.

[0280] For example, a kit may include at least one cassette and aplurality of electrode wicks. The kit may optionally additionallycomprise sealing tape suitable for sealing entries 14 and 16 duringrehydration, and/or may optionally contain a plurality of IPG strips,either with identical pH ranges or differing pH ranges.

[0281] Kits of the present invention may also, optionally, include anyone or more of separately containerized carrier ampholytes, cathodebuffer, anode buffer, stains (such as silver stains or colloidal bluestains)—either in liquid form, at 1× use concentration or higherconcentration for further dilution, or in dry form to be reconstitutedwith water of suitable quality—protein standards, typically inadmixture, or polyacrylamide gels suitable for performing a seconddimension of separation following isoelectric focusing, such as aTris-Glycine ZOOM® gel (Invitrogen, Carlsbad, Calif.).

EXAMPLE 1 Determination of Channel Tolerances

[0282] Three cassettes were manufactured by machining six parallelchannels each into form-retaining plastic slabs, with geometryessentially as shown in FIG. 1A. The six channels of the first cassetteall were 0.77 mm in depth, with two channels 4.09 mm in width, twochannels 0.65 mm in width, and two channels 3.35 mm in width. The sixchannels of the second cassette all were 0.65 mm in depth, with twochannels 4.09 mm in width, two channels 0.65 mm in width, and twochannels 3.35 mm in width. The six channels of the third cassette allwere 0.57 mm in depth, with two channels 4.09 mm in width, two channels0.65 mm in width, and two channels 3.35 mm in width. The channels wererendered fluidly enclosing except at terminal entries by application ofa flexible laminate cover to each of the three cassettes.

[0283] Serva IEF standard, 5 μL, (catalogue no. 39212-01, ServaElectrophoresis GmbH, Heidelberg, Germany) was mixed with 120 μL ofrehydration buffer of the following composition: 8.0 M urea, 0.5%ampholytes (3-10 IPG buffer, cat. no. 17-6001-11, Amersham Biosciences),2.0% (w/v) CHAPS, 20 mM DTT, 0.0025% (w/v) bromphenol blue. The solutionwas pipetted into each channel of the three cassettes, with the cassettepositioned horizontally.

[0284] An Immobiline DryStrip 3-10 7 cm gel (Amersham Biosciences,Piscataway, N.J., USA) was inserted into each channel. The channelentries were occluded with cover tape and the strips allowed torehydrate for 8 hours. Cover tape was removed, as were loading wells ifpresent.

[0285] A filter paper wick dampened with water was placed in contactwith the extreme ends of the gel portion of the strip at the terminalentries.

[0286] Electrodes were contacted to the wick at the anodic and cathodicends of the cassette and a voltage applied in three steps according tothe following protocol: 250 volts for 15 minutes, ramp from 250-3500volts for 1 hour and 30 minutes, and 3500 volts for 1 hour. Current waslimited to 1 mA and power to 4 watts in all three steps.

[0287] Strips were removed, stained with Coomassie blue stain, aligned,and photographed.

[0288] Results shown in FIG. 8 indicate that even the largest channel,4.09 mm in width and 0.77 mm in depth, permitted adequate focusing ofthe Serva IEF standard (left-most lane) in strips with nominal width of3 mm and depth of 0.5 mm.

EXAMPLE 2 Comparison of Electrical Properties Using a Ramped VoltageProfile

[0289] This experiment compares the actual current flow through IPGstrips run (i) in a commercial horizontal IPG electrophoresis apparatus(Multiphor™ flatbed system, Amersham Biosciences, Inc., Piscataway,N.J.) and (ii) in the cassette of the present invention using the buffercore of the present invention for vertical electrophoresis (ZOOM®IPGRunner™), using a ramped voltage power profile. For furthercomparison, strips from two vendors were used, Amersham Biosciences,Inc. (“AP strips”) and Invitrogen Corporation (“INV strips”).

Equipment and Materials

[0290] 1. Power Supplies: Novex Model 35-40. A quantity of four of theseunits were used, one for each of the four individual experimentalsetups.

[0291] a. Voltage/Time profile: The same ramped voltage profile was setfor each of the four systems. The profile set was:

[0292] 0→250V over 15 minutes

[0293] 250V→3500 V over 1.5 hours

[0294] 3500V for 2 hours

[0295] 2. Digital Multi Meters (DMMs) for measurement:

[0296] Two were used, both were capable of microampere measurements. TheDMMs were used in current-measurement mode. They were set up in serieswith the ground-side power cable between the apparatus and the powersupply.

[0297] a. Fluke 87 series III

[0298] b. Fluke 73 series III

[0299] 3. Multiphor™

[0300] a. Wick solution: 0.5 ml ultra-pure water

[0301] b. Rehydration: 0.125 ml/strip 4. ZOOM® IPGRunner™

[0302] a. Wick solution: 0.75 ml de-ionized water

[0303] b. Rehydration: 0.155 ml/strip

[0304] 5. Strips

[0305] a. Invitrogen 4-7 pH, Lot #100248

[0306] b. AP Biotech 4-7 pH, Lot #287374

[0307] 6. Sample in Rehydration Solution:

[0308] a. second spin lysate from E. Coli (0.002 ml per 1 ml rehydrationsolution)

[0309] b. after adding lysate, centrifuged 15 min at 14,000 rpm

[0310] c. pH 4-7 ZOOMlytes (Invitrogen Corp.)

Results

[0311] TABLE 1 Actual Currents Using Multiphor ™ and ZOOM ® IPGRunner ™CURRENT, mA, as measured (VOLTAGE, V, as reported by Power Supply)Multiphor ™ ZOOM ® IPG Runner ™ #1 #2 #1 #2 Elapsed w/AP w/Inv w/APw/Inv Time (h:m) strips strips strips strips 0:15 .12 .10 .43 .42 0:20.11 .14 .38 .45 ˜0:25    .145  .185 .33 .53 ˜0:40    .205 Nr .17 .19(1250 V) (1170 V) ˜0:50    .088 Nr nr .20 (1710 V) (1515 V) ˜1:00   .097  .100 .24 .24 (2100 V) (1908 V) (2108 V) (1908 V) ˜1:15   Nr  .097nr .29 (2500 V) (2500 V) Time @ — — — — 3500 V: 0:05 .15 .12 .39 .39(3500 V) (3500 V) (3500 V) (3500 V) 0:10 .15 .12   .45 ¹ .38 (3500 V)(3500 V) (3500 V) 0:15 .15 .12 .46 .35 (3500 V) (3500 V) (3500 V) (3500V) 0:20 .15 .11 .42 .32 (3500 V) (3500 V) (3500 V) (3500 V) 0:30 .15 .11.36 .30 (3500 V) (3500 V) (3500 V) (3500 V) 1:15 .15 .11 .27 .65/.28 ²(3500 V) (3500 V) (3500 V) 1:40 .20 .20 (3500 V) (3500 V) #(100 mssampling) was .82 mA during the burn.

[0312] Results are plotted in FIG. 9. As can be seen from both Table 1and FIG. 9, there is substantially higher current flow through the IPGstrips using the cassette and buffer core of the present invention ascompared to a flatbed device, at all times during the run and witheither brand of IPG strip.

[0313] Table 2 presents “volt-hour” ratios for the two types of devicesduring the constant 3500 Volt period of the power cycle program.

[0314] The “volt-hour” ratios are calculated from the measuredcurrents—given identity in resistance through the IPG strips, measuredcurrent-hour ratios are equivalent to volt-hour ratios. Ratios arecalculated as IPGRunner™ (IPGRunner) “volt-hours” divided Multiphor™(MP) “volt-hours”. TABLE 2 “Volt-hour” Ratios V-hour ratios @ 3500 V:IPGRunner/MP Time AP strips INV strips 0:05:00 2.7 3.3 0:10:00 3.0 3.20:15:00 3.1 2.9 0:20:00 2.8 2.9 0:30:00 2.4 2.7 1:15:00 1.8 2.5 Average2.6 2.9

[0315] Table 2 demonstrates that the cassette and buffer core of thepresent invention are far more efficient at transferring power to thegels than is the flatbed Multiphor™, despite apparent identity in thenominal volt-hours calculated by multiplying power supply voltage bytime.

[0316] During the first, low voltage, stage of the profile, the cassetteand buffer core of the present invention pushes up to 4 times the amountof current through the strips (both AP and INV strips) than theMultiphor™ does. There were visible differences in the velocity andshape of the dye fronts between the two apparatuses during this stage.

[0317] During the high voltage focusing plateau, set at 3500 V in thisexperiment, the cassette and buffer core of the present inventiondelivered an average of approximately 2.6 times and 2.9 times morecurrent to the AP and INV strips, respectively, than did the Multiphor™.

[0318] Using a ramped voltage profile, lower voltages and times may beused to achieve focusing with the cassette and buffer core of thepresent invention than has been used for prior flat bed devices. Lowervoltages for shorter periods are more convenient, require lesssophisticated (and expensive) power supplies, obviate the need forcurrent limiting power supplies, and should lead to less frequent arcingand thus burning of the strips. Optimal voltages and times will, asbefore, depend in part on sample osmolality and electrolyteconcentration.

EXAMPLE 3 Electrical Properties Using a Stepped Voltage Profile

[0319] Four full cassettes with six strips each of various pH ranges,each strip loaded with a standard sample, were run in two ZOOM®IPGRunner™ systems (cassette, buffer core, tank) of the presentinvention in order to evaluate the effectiveness of a voltage stepprofile without current or watt limits.

Equipment and Materials

[0320] 1. Power Supplies: Novex Model 35-40. A quantity of two of theseunits were used, one for each of the two individual experimental setups.

[0321] a. Voltage/Time profile: The same voltage profile was set on bothsystems, as set forth in Table 3, below.

[0322] 2. Digital Multi Meters for measurement. Two were used, bothcapable of micro Amp measurements. The DMMs were used incurrent-measurement mode. They were set up in series with theground-side power cable between the apparatus and the power supply.

[0323] a. Fluke 87 series III

[0324] b. Fluke 73 series III TABLE 3 Stepped Voltage Profile VoltageDuration Time, accumulated 175 15 min 0:00-0:15 500 15 min 0:15-0:30 75015 min 0:30-0:45 2000 30 min 0:45-1:15

Results

[0325] Results are summarized in Table 4 and plotted in FIG. 10. TABLE 4Measured Current and Nominal Voltages, Step Profile CURRENT, mA, asmeasured (VOLTAGE, V, as reported by Power Supply) ZOOM ® IPG Runner ™ZOOM ® IPG Runner ™ Elapsed (power unit #1) (power unit #2) Time (h:m)Current Voltage Current Voltage 0:00  .03   0 0.03   0 0:01  .68  175 .72  175 0:02  .70  175  .74  175 0:03  .71  175  .74  175 0:04  .70 175  .72  175 0:05  .68  175  .69  175 0:10  .55  175  .55  175 0:15 .43/1.21  175/500  .42/1.20  175/500 0:16 1.19  500 1.15  500 0:17 1.10 500 1.15  500 0:18 1.06  500 1.13  500 0:19 1.00  500 1.08  500 0:20 .93  500 1.05  500 0:25  .59  500  .59  500 0:30  .40/.60  500/750 .44/.66  500/750 0:31  .57  750  .63  750 0:32  .54  750  .60  750 0:33 .51  750  .55  750 0:34  .48  750  .51  750 0:35  .45  750  .46  7500:40  .30  750  .31  750 0:45  .24/.67  750/2000  .26/.72  750/2000 0:46 .68 2000  .71 2000 0:47  .68 2000  .71 2000 0:48  .65 2000  .68 20000:49  .63 2000  .66 2000 0:50  .61 2000  .64 2000 0:51  .58 2000  .622000 0:52  .57 2000  .60 2000 0:53  .55 2000  .59 2000 0:54  .54 2000 .59 2000 0:55  .53 2000  .58 2000 1:00  .52 2000  .55 2000 1:05  .512000  .51 2000 1:10  .50 2000  .50 2000 1:15  .48/0.01 2000/0.0  .492000 1:45 finished finished 2000/0

[0326] The stepped voltage profile resulted in no burning of any of the24 strips.

[0327] The 500 volt step resulted in 99 μamps per strip for less than aminute, but was over 70 μamps for approximately 5 minutes; however, 500volts is a significantly lower voltage than the final focusing voltage,and the conductivity of the sample at this low voltage is not likely tocause any arcing or burning. No arcing or burning was seen.

[0328] This experiment demonstrates that an inexpensive unregulatedpower supply capable of only “step” profiles can be used to focussamples using a variety of IPG strips with the cassette and buffer core(ZOOM® IPGRunner™ system) of the present invention, without burning ofthe strips, and with high quality isoelectric focusing. The powersupplies are programmable but do not provide current limits in themicroampere range.

[0329] Additionally, the tested profile appears to provide an excellentbalance by placing the higher current loads down in the lower voltageregions. Optimal voltages and times will, however, depend in part onsample osmolality and electrolyte concentration.

[0330] All patents and publications cited in this specification areherein incorporated by reference as if each had specifically andindividually been incorporated by reference herein. Although theforegoing invention has been described in some detail by way ofillustration and example, it will be readily apparent to those ofordinary skill in the art, in light of the teachings herein, thatcertain changes and modifications may be made thereto without departingfrom the spirit or scope of the appended claims, which, along with theirfull range of equivalents, alone define the scope of invention.

What is claimed is:
 1. A system for low resistance electrophoresis ofanalyte samples in prior-cast, hydratable separation media strips,comprising: means for enclosing a plurality of said strips, saidenclosing means permitting spaced electrical communication separatelywith each of said enclosed strips through respective first and secondentries; and means responsive to an external compressive force foreffecting spaced electrical communication by a single anode and singlecathode simultaneously with each of said enclosed strips, wherein saidelectrical communication means is capable of distributing an externalcompressive force to urge said anode and said cathode toward saidenclosing means with greater pressure at said first and second entriesthan elsewhere on said enclosing means.
 2. The system of claim 1,wherein said enclosing means is capable of hydratingly lodging saidstrips there within.
 3. The system of claim 2, wherein said electricalcommunication means comprises: an anode support; a cathode support; ananode; and a cathode; wherein said supports discontinuously distributean external compressive force to said anode and cathode to urge saidanode and said cathode toward said enclosing means with greater pressureat said first and second entries than elsewhere on said enclosing means.4. The system of claim 2, wherein said anode support makes discontinuouscontact with said anode and said cathode support makes discontinuouscontact with said cathode.
 5. The system of claim 1, capable ofelectrophoretic separation in said strips with application of 3000 orfewer volts.
 6. The system of claim 5, capable of electrophoreticseparation in said strips with application of 1500 or fewer volts. 7.The system of claim 6, capable of electrophoretic separation in saidstrips with application of 500 or fewer volts.
 8. The system of claim 1,capable of electrophoretic separation in said strips with application offewer than 2000 volt-hours.
 9. The system of claim 8, capable ofelectrophoretic separation in said strips with application of fewer than1500 volt-hours.
 10. The system of claim 1, capable of electrophoreticseparation in said strips in fewer than 6 hours.
 11. The system of claim10, capable of electrophoretic separation in said strips in fewer than 5hours.
 12. The system of claim 10, capable of electrophoretic separationin said strips in fewer than 4 hours.
 13. A method for low resistanceelectrophoresis of analyte samples in prior-cast, hydratable separationmedia strips, the method comprising: hydratingly lodging at least onestrip within an enclosing member that permits separate, spaced,electrical communication with each of a plurality of enclosed stripsthrough respective first and second entries; applying a samplecontaining protein analytes to said at least one strip; forcibly urgingan anode and a cathode toward said enclosing member to effectsimultaneous spaced electrical communication with each of said strips,wherein the force urging said anode and said cathode toward saidenclosing member is distributed to create greater contact pressure atsaid first and second entries than elsewhere on said enclosing member;and then applying electrical potentials to said anode and said cathodeat a potential difference and for a time sufficient to effectelectrophoretic separation of analytes in said at least one strip. 14.The method of claim 13, wherein said sample is applied during lodging ofsaid strip in said enclosing member.
 15. The method of claim 13, whereinsaid applied potential difference is 3000 or fewer volts.
 16. The methodof claim 15, wherein said applied potential difference is 1500 or fewervolts.
 17. The method of claim 16, wherein said applied potentialdifference is 500 or fewer volts.
 18. The method of claim 13, whereinsaid potential difference is applied for fewer than 6 hours.
 19. Themethod of claim 18, wherein said potential difference is applied forfewer than 5 hours.
 20. The method of claim 19, wherein said potentialdifference is applied for fewer than 4 hours.
 21. The method of claim20, wherein said potential difference is applied for fewer than 3 hours.22. The method of claim 13, wherein said potential difference is appliedfor fewer than 2000 volt-hours.
 23. The method of claim 22, wherein saidpotential difference is applied for fewer than 1500 volt-hours.
 24. Themethod of claim 23, wherein said potential difference is applied forfewer than 1400 volt-hours.
 25. The method of claim 13, wherein saidpotential difference is applied in a plurality of ramped voltage steps.26. The method of claim 13, wherein said potential difference is appliedin a plurality of stepped voltage steps.
 27. The method of claim 13,wherein said potential difference is applied at a constant voltagelevel.
 28. The method of claim 13, wherein said strip is an IPG strip.29. A device for forcibly urging an anode and a cathode intosimultaneous spaced electrical communication with a plurality ofprior-cast hydratable separation media strips enclosed within means thatpermit spaced electrical communication separately with each of theenclosed strips through respective first and second entries, comprising:a substantially inflexible frame; an anode support; a cathode support;an anode; and a a cathode; wherein said anode support and said cathodesupport are spacedly fixed to said frame and are capable of distributingan external compressive force respectively to said cathode and saidanode to urge said cathode and said anode toward said strips withgreater contact pressure at said first and second entries than elsewhereon said enclosing means.
 30. The device of claim 28, wherein said anodesupport makes intermittent contact with said anode and said cathodesupport makes intermittent contact with said cathode.
 31. In a method ofelectrophoresis using prior-cast, hydratable, separation media strips,the improvement comprising: spacedly contacting said strips with ananode and cathode with resistance between power supply and gelsufficiently low as to permit electrophoretic separation with a maximumapplied voltage of no more than 3000 volts.
 32. The method of claim 31,wherein said maximum applied voltage is no more than 1500 volts.
 33. Themethod of claim 32, wherein said maximum applied voltage is no more than500 volts.
 34. The method of claim 31, wherein said strip is animmobilized pH gradient (IPG) strip, and said resistance is sufficientlylow as to permit isoelectric focusing with application of fewer than2000 nominal volt-hours.
 35. The method of claim 34, wherein saidresistance is sufficiently low as to permit isoelectric focusing withapplication of fewer than 1500 nominal volt-hours.